Biomarkers

ABSTRACT

The invention provides binding agents and assays tor insulin signal peptide. The agents and assays are useful in methods tor predicting, diagnosing, assessing or monitoring acute cardiac disorders, glucose handling disorders and diabetes in a subject. Also provided are nucleotides, polypeptides, and kits useful in the methods of the invention.

FIELD OF THE INVENTION

This invention relates to insulin signal peptide (INS-SP) and its use inthe prognosis, diagnosis and monitoring of biological events disordersor states which result in release of the marker into the circulation.Such events including glucose handling disorders, diabetes andassociated conditions such as cardiovascular disease, particularly acutecardiac disorders.

BACKGROUND

Diabetes Mellitis is a metabolic disorder characterized by deficienciesin insulin secretion, insulin action or both. These deficiencies resultin chronic hyperglycemia. Diabetes also has a number of associatedconditions including obesity and an increased risk of cardiac disordersincluding cardiovascular disease. Diabetes affects over 170 millionpeople worldwide, and is expected to double in the next twenty years.

Diabetes is divided into two types known as Type 1 diabetes and Type 2diabetes. Type 1 diabetes is an autoimmune related disorder where theimmune system of the individual acts to destroy the beta cells of thepancreas. Individuals with Type 1 diabetes are generally insulindependent. They exhibit limited insulin secretion, if any.

Type 2 diabetes is the most common form, accounting for 90 to 95% ofcases. The majority, of Type 2 diabetics are not insulin dependent, butexhibit insulin secretion and insulin action deficiencies leading tohyperglycemia. The hyperglycemia is often mild with symptoms difficultto recognise. As a result, many Type 2 diabetics go undiagnosed for manyyears. At any given time it is estimated that 10 to 15% of thepopulation may be at risk of developing Type 2 diabetes, but areundiagnosed.

Diabetes is most commonly diagnosed based on the oral glucose tolerancetest which assesses glucose handling. Individuals are given a glucosedrink after overnight fasting to test their tolerance for glucose. Thetest takes several hours to measure responses. Unfortunately, theglucose tolerance test and fasting insulin level test suffer from a lackof sensitivity, and false positives which limit their usefulness asprognostic indicators of diabetes.

Diabetes is a significant risk factor for cardiovascular disease,increasing the risk of a cardiac event by two to three times. Despitethe recognised need for diagnostic and prognostic tools for assessingthe risk of an individual developing diabetes, precursor glucosehandling disorders, and associated conditions such as cardiovasculardisease, no simple and accurate tests are available.

It is an object of the present invention to go some way towards fillingthese needs and/or to at least provide the public with a useful choice.

Early diagnosis and ongoing assessments of diabetes and precursorglucose handling disorders, or any other form of dysglycemia ordysinsulinemia, are important not only for the management thereofdiabetes, but also for managing associated conditions, such ascardiovascular disease. In addition to providing early detection methodsfor conditions, diseases associated with dysglycemia or dysinsulinemia,for example, the present invention also has broader applicationsincluding in the cardiovascular area.

Acute cardiac disorders including acute coronary syndromes (ACS)encompass a wide spectrum of cardiac ischemic events ranging fromunstable angina through to acute myocardial infarction (AMI). AMIpresents as the most serious of these events and therefore requiresrapid and accurate diagnosis. Patients who present with two or more ofthe described features (a history of ischemic chest discomfort,evolutionary changes on serial electrocardiogram (ECG) traces and a riseand fall in plasma cardiac biomarkers) are clearly identified asundergoing AMI.²⁶ However, a significant proportion of patients(40%-50%) who present with suspected AMI do not have serial changes onECG, or typical symptoms thus placing heavy emphasis on circulatingbiomarker concentrations for accurate diagnosis.^(26,27)

Accurate early diagnosis of myocardial infarction facilitates promptintroduction of reperfusion treatment, including effective percutaneousor thrombolytic revascularisation and adjunctive anticoagulant andanti-platelet therapy. Such treatments are progressively less effectiveat reducing mortality and morbidity with each hour of delay in diagnosisand management.²⁻⁴ Given the need for accelerated decision-making inthis clinical situation, there is a need for identification ofcirculating biomarkers providing an early and specific diagnosis ofacute cardiac disorders, particularly AMI, for example.

Indeed current clinical guidelines highlight the importance of biomarkermeasurement in the identification of myocardial infarction and acutecoronary syndromes.²⁶ A number of biomarkers have been proposed for thispurpose, including creatine kinase-MB (CK-MB), troponin T (TnT),troponin I (TnI) BNP, N-BNP (also known as NP-BNP), BNP signal peptide(BNP-SP) and myoglobin, but there are limitations to their use. Time todetectable or abnormal elevation of plasma cardiac biomarkers can be 6hours (myoglobin, CK-MB) to 12 hours (TnT, TnI, BNP, N-BNP) with peaklevels not occurring until 24-48 hours after onset of injury, imposing awindow of delay upon precise diagnosis and treatment.¹⁻⁴ Furthermore,both myoglobin and CK-MB are non-specific and can be secreted fromextra-cardiac sources, especially during trauma or surgery.¹

The long term diagnostic/predictive powers of the known markerstherefore lack the accompanying power of a specific marker providingearly specific diagnosis of acute cardiac disorders such as acutecardiac injury within the first few hours of clinical presentation. Aneed thereof still exists for early markers.

It is a further object of the present invention to provide an earlymarker of acute cardiac disorders, and/or to at least provide the publicwith a useful choice.

SUMMARY OF THE INVENTION

Human insulin signal peptide (INS-SP) is a 24 amino acid peptide cleavedfrom proinsulin (preproinsulin) (1-110) SEQ ID NO:1. Processing of humaninsulin is shown in FIG. 4. INS-SP (1-24) is shown separately in SEQ IDNO:14.

The applicants have found for the first time that INS-SP and fragmentsthereof are released into the circulation. Useful circulating biomarkersare identified and provided. Previously it was thought that INS-SP wasonly ever produced intracellularly.²⁵

Based on this finding, the applicants provide in one aspect of theinvention a method for predicting, diagnosing, assessing or monitoring abiological event or disorder in a subject wherein the event or disordercorrelates with the release of one or more INS-SP biomarkers into thecirculation, the method comprising measuring the level of one or moreINS-SP biomarkers in a sample taken or derived from the subject, andanalyzing the level in conjunction with a respective reference valuerange for said one or more biomarkers.

On one embodiment, the INS-SP biomarker is an INS-SP. In anotherembodiment, the INS-SP biomarker is an INS-SP fragment. In one preferredembodiment, the INS-SP fragment is human INS-SP (1-9) (SEQ ID NO:16). Inanother preferred embodiment, the INS-SP fragment is human INS-SP(15-24) SEQ ID NO:18. In other preferred embodiments, the INS-SPfragment comprises human INS-SP (1-9) (SEQ ID NO:16) or human INS-SP(15-24) SEQ ID NO:18.

In another embodiment, the method comprises comparing the level of anINS-SP biomarker, preferably an INS-SP fragment, in one or more samplestaken or derived from the subject with the INS-SP biomarker level from acontrol wherein a deviation in the measured level from the control levelis indicative of a biological event or disorder.

In a diabetic subject, such as a type 1 diabetic subject, or in asubject with another dysglycemia resulting from depressed insulinsecretion levels, the level of insulin will be different from normal, aswill INS-SP and/or INS-SP fragment levels. This finding indicates thatINS-SP is useful as a marker for such conditions. Depending on theinsulin state of the subject, INS-SP biomarker levels in the subjectwill be higher or lower than normal.

In a diabetic subject, such as a type 2 diabetic subject, or in asubject with another dysinsulinemia, the level of insulin will bedifferent than normal, as will INS-SP and/or INS-SP fragment levels.This finding indicates that INS-SP and/or INS-SP fragments are alsouseful as a marker for such conditions, as well as in otherhyperinsulinemic states such as metabolic syndrome. Depending on theinsulin state of the subject, INS-SP biomarker levels in the subjectwill be higher or lower than normal.

Accordingly, in another aspect the present invention provides a methodfor predicting, diagnosing, assessing or monitoring diabetes or diabeticpotential, as well as other conditions characterized by dysglycemiaand/or dysinsulinemia, the method comprising measuring the level of oneor more INS-SP biomarkers in a sample taken or derived from the subject,and analysing the level in conjunction with a respective reference valuerange for said one or more biomarkers.

In another embodiment, the method comprises comparing the level of anINS-SP biomarker, preferably an INS-SP fragment, in one or more samplestaken or derived from the subject with the INS-SP biomarker level from acontrol wherein a measured level of INS-SP that deviates from thecontrol level is indicative of diabetes or a predisposition to diabetes,or another condition associated with dysglycemia and/or dysinsulinemia.In one embodiment the INS-SP biomarker level may be lower than thecontrol.

The invention also provides a method of assessing glucose handling in asubject, the method comprising:

-   -   (a) measuring the level of INS-SP biomarker, preferably an        INS-SP fragment, in a subject after administration of glucose;        and    -   (b) comparing the level of said INS-SP with the INS-SP from a        control,    -   wherein a deviation in the measured level of INS-SP from the        control level is indicative of a glucose handling disorder.

The applicants have also surprisingly discovered that the circulatingconcentration of INS-SP biomarkers are highest in the first few hoursfollowing onset of, or at clinical presentation with suspected acutecoronary syndromes (ACS). Peaks are in the order of five to fifteentimes higher, than normal control populations in these first hours.

Accordingly, in a further aspect the present invention provides a methodfor predicting, diagnosing or monitoring an acute cardiac disorder (ACD)in a subject, the method comprising measuring the level of an INS-SPbiomarker, preferably an INS-SP fragment, in a biological sample fromthe subject and comparing the level of said INS-SP biomarker with theINS-SP and/or INS-SP fragment level from a control or reference value orvalue range wherein a measured level of the INS-SP biomarker higher thanthe control level, or a predetermined reference value or value range, isindicative of ACD.

The invention also provides a method for monitoring a response totreatment of an acute cardiac disorder (ACD) in a subject, the methodcomprising measuring the level of an INS-SP biomarker, preferably anINS-SP fragment, in a biological sample taken or derived from thesubject and comparing the level of said INS-SP biomarker with the INS-SPbiomarker level from a control or reference value or value range,wherein a change in the measured level of the INS-SP biomarker from thecontrol level, or a predetermined reference value or value range, isindicative of a response to the treatment.

In another aspect, the invention also provides a method for predicting,diagnosing or monitoring a cardiac transplant rejection episode in asubject, the method comprising measuring the level of an INS-SPbiomarker, preferably an INS-SP fragment, in a biological sample takenor derived from a subject after heart transplant and comparing the levelof said INS-SP biomarker with the INS-SP biomarker level from a controlor reference value or value range, wherein a measured level of theINS-SP biomarker higher than the control level, or a predeterminedreference value or value range, is indicative of transplant rejection ora transplant rejection episode.

The invention also provides a method of distinguishing between apulmonary disorder and an acute cardiac disorder (ACD) in a subject, themethod comprising measuring the level of an INS-SP biomarker, preferablyan INS-SP fragment, in a biological sample taken or derived from thesubject and comparing the level of said INS-SP biomarker with the INS-SPbiomarker level from a control, or a predetermined reference value orvalue range, wherein a measured level of the INS-SP biomarker higherthan the control level, or a predetermined reference value or valuerange, is indicative of ACD.

The invention also provides a method for predicting, diagnosing ormonitoring an acute cardiac disorder (ACD), cardiac transplantrejection, or ACD/pulmonary disorder in a subject, the method comprisingmeasuring the level of an INS-SP biomarker, preferably an INS-SPfragment, in a biological sample taken or derived from the subjectwithin about the first two hours of onset of, or clinical presentationwith ACD, cardiac transplant rejection or ACD/pulmonary disorder,comparing the measured level of the INS-SP biomarker with the INS-SPbiomarker level from a control, or reference value or value range,wherein a measured level of the INS-SP biomarker higher than the controllevel, or a predetermined reference value or value range, is indicativeof ACD or cardiac transplant rejection, or a transplant rejectionepisode.

In a broader embodiment the applicant's findings can be used to predict,diagnose, assess or monitor any event in which INS-SP, or an INS-SPfragment, is released into the circulation.

In one embodiment of the cardiac methods of the invention the INS-SPbiomarker level is measured one or more times on samples (or samplederivatives) taken from a subject within about six hours, about fourhours, about two hours, about one hour, about 30 minutes, or withinabout 15 minutes of presentation with the disorder, or its occurance.Single or multiple INS-SP biomarker measurements within six hours, fourhours, two hours, one hour, one-half hour, and one-quarter hour areincluded within the invention. INS-SP biomarker measurements oradditional INS-SP biomarker measurements on samples subsequently takenor derived from a subject following six hours are also included.

In one embodiment, the methods of the invention are in vitro methods.

In one embodiment, the sample is blood, saliva, interstitial fluid,plasma, urine, serum or heart tissue. In one preferred embodiment, thesample is blood or plasma.

In one embodiment, the measuring step comprises detecting bindingbetween INS-SP and a binding agent that selectively binds INS-SP. Themeasuring step in one embodiment comprises:

-   -   (a) binding the INS-SP biomarker with a binding agent; and    -   (b) measuring the level of bound INS-SP biomarker.

The binding agent in one embodiment is an antibody or antigen-bindingfragment thereof. Most commonly, the antibody is a monoclonal,polyclonal, bispecific, chimeric or humanized antibody. In oneembodiment the antibody is a monoclonal antibody.

In another embodiment, the levels of an INS-SP biomarker are measuredusing mass spectroscopy.

The INS-SP biomarker which is bound or detected by the antibody is thefull length human INS-SP molecule (SEQ ID NO:14) or an antigenic variantor fragment thereof. In one embodiment, the fragment is at least fourcontiguous amino acids in length. In another embodiment the fragmentthat is bound or detected is human INS-SP (1-9) (SEQ ID NO:16), INS-SP(15-24) SEQ ID NO:18. The antibody may bind the N-terminus or theC-terminus of the INS-SP or the INS-SP fragment.

Specific antigenic peptides which the binding agent selectively bindsinclude human INS-SP (1-9) (SEQ ID NO:16), INS-SP (15-24) SEQ ID NO:18,or antigenic-binding fragments, or variants thereof.

Binding of the INS-SP biomarker in one embodiment is measured usingantibodies or antibody fragments that are immobilised on a solid phase.

Levels of an INS-SP biomarker may usefully be measured with an assayselected from RIA, ELISA, fluoroimmunoassay, immunofluorometric assay,mass spectrometry and immunoradiometric assay.

Accordingly, the invention also provides an assay for an INS-SPbiomarker in a biological sample from a subject, the assay comprisingdetecting and measuring the level of the INS-SP biomarker in the sampleor sample derivative using any known methods.

The invention also provides an assay for an INS-SP biomarker comprising:

-   -   (a) binding one or more INS-SP biomarkers from a sample; and    -   (b) measuring the level of bound INS-SP biomarker.

The INS-SP biomarker may be bound using an INS-SP biomarker-bindingagent of the invention.

The invention also provides an INS-SP biomarker assay for use inpredicting, diagnosing, assessing or monitoring a biological event ordisorder in a subject.

In one embodiment, the assay is an in vitro assay.

The dysglycemia-related methods of the invention may further comprisemeasuring the level of one or more non-INS-SP/INS-SP fragment markersof, for example, diabetes and comparing the levels against marker levelsfrom a control wherein a deviation in the measured level from thecontrol level of non-INS-SP marker, together with a measured level ofINS-SP which deviates from or is lower than the control level of INS-SPis predictive or diagnostic of, for example, diabetes or can be used tomonitor diabetes, for example. Non-INS-SP/INS-SP fragment markers fordiabetes may include glucose, insulin, lactate and triglyceride or fattyacid levels or markers thereof. Other markers include HbA1C andfructoseamine.

The cardiac-related methods of the invention may further comprisemeasuring the level of one or more non-INS-SP or non-INS-SP fragmentmarkers of said ACD, or cardiac transplant rejection, or ACD/pulmonarydisorder and comparing the levels against marker levels from a controlor reference value or value range, wherein a deviation in the measuredlevel from the control or reference level of the non-INS-SP marker,together with a measured level of the INS-SP biomarker which is higherthan a control or reference INS-SP biomarker level, is predictive ordiagnostic of the ACD, or can be used to assess or monitor said ACD(including cardiac transplant rejection) or ACD/pulmonary disorder.

Markers for use in the context of acute coronary syndrome includetroponin, troponin T, troponin I, creatine kinase MB, myoglobin, BNP,NT-BNP, BNP-SP, BNP-SP fragments, ANP, ANP-SP, ANP-SP fragments, LDH,aspartate aminotransferase, heart specific fatty acid binding protein(H-FABP), ischemia modified albumin, endothelin, adrenomedullin andangiotensin II.

In another aspect, the present invention also provides an INS-SPbiomarker binding agent. In one embodiment, the INS-SP biomarker bindingagent of the invention binds or detects:

-   -   (a) INS-SP (1-24) SEQ ID NO:14;    -   (b) INS-SP (1-9) SEQ ID NO:16;    -   (c) INS-SP (15-24) SEQ ID NO:18;    -   (d) an amino acid sequence encoded by a nucleotide sequence        selected from SEQ ID NO: 15, SEQ ID NO:17 and SEQ ID NO:19; or    -   (e) a variant or fragment of any one of (a) to (d).

The binding agent is useful in predicting, diagnosing, assessing ormonitoring a biological event or disorder which correlates with therelease of an INS-SP or INS-SP fragment into the circulation. Suchevents or disorders include diabetes, glucose handling disorders, andacute cardiac disorder (ACD) in a subject.

In one embodiment, the binding agent is an anti-INS-SP antibody or ananti-INS-SP fragment antibody or an antigen-binding fragment of either.

The invention also provides an anti-INS-SP biomarker antibody orantigen-binding fragment thereof which binds:

-   -   (a) INS-SP 1-24 (SEQ ID NO:14);    -   (b) INS-SP 1-9 (SEQ ID NO:16);    -   (c) INS-SP 15-24 (SEQ ID NO:18); and    -   (d) an amino acid sequence encoded by a nucleotide sequence        selected from SEQ ID NO:15, SEQ ID NO:17 and SEQ ID NO:19; or    -   (e) a variant or fragment of any one of (a) to (d).

The antibody may be a monoclonal, polyclonal, bispecific, chimeric, orhumanized antibody, or binding fragments or constructs of either.

The invention is also directed to the use of an INS-SP biomarker bindingagent in the manufacture of an INS-SP biomarker assay for assessing abiological event or disorder in a subject, or to the use of an INS-SPbiomarker binding agent in the manufacture of a prognostic, diagnostic,assessment or monitoring tool for a biological event or disorder in asubject. In one embodiment, the event or disorder correlates with therelease of INS-SP and/or an INS-SP fragment into the circulationincluding from a glucose handling disorder, diabetes, or an acutecardiac disorder (ACD).

The invention also relates to the use of an antibody or antigen-bindingfragment of the invention in the manufacture of a prognostic,diagnostic, assessment or monitoring tool for a biological event whichcorrelates with the release of INS-SP and/or an INS-SP fragment into thecirculation including a glucose handling disorder, diabetes, acutecardiac disorder (ACD), cardiac transplant rejection or an ACD/pulmonarydisorder in a subject.

In one embodiment the prognostic, diagnostic or monitoring tool iscalibrated to measure INS-SP levels in the range of from about 0.1 toabout 500 pmol/L, or about 1 to about 300 pmol/L, or about 10 to about250 pmol/L.

In another aspect, the invention provides a kit for predicting,diagnosing or monitoring a biological event in a subject, the kitcomprising an INS-SP biomarker binding agent of the invention.

In one embodiment the kit is calibrated to measure INS-SP biomarkerlevels in the range of about 0.1 to about 500 pmol/L, about 1 to about300 pmol/L, or about 10 to about 250 pmol/L.

In one embodiment the kit also includes instructions for predicting,diagnosing, assessing or monitoring a biological event or disorderincluding, for example, diabetes or an ACD in a subject, from the INS-SPbiomarker level measured in a sample or derivative of a sample andcomparing the measured level to a control or reference level. A measuredINS-SP biomarker level which deviates from the control or referencelevel is indicative of a biological event or disorder, such as, forexample, a glucose handling disorder, diabetes or ACD (includingtransplant rejection).

In another aspect, the invention relates to a nucleic acid moleculeencoding an INS-SP fragment of the invention wherein said nucleic acidis selected from

-   -   (a) SEQ ID NO:17 or a variant or fragment thereof;    -   (b) SEQ ID NO:19 or a variant or fragment thereof;    -   (c) a sequence which has at least 70%, 75%, 80%, 85%, 90%, 95%,        or 99% sequence identity to (a) or (b);    -   (d) a sequence of at least 10 nucleotides in length, capable of        hybridising under stringent conditions to any one of (a) to (c);        and    -   (e) a complement of any one of (a) to (d)    -   with the proviso that the sequence is not SEQ ID NO:15.

In one embodiment, the INS-SP fragment encoded by the nucleic acidmolecule is INS-SP (1-9) SEQ ID NO:16, or INS-SP (15-24) SEQ ID NO:18.

The invention also provides a genetic construct comprising a nucleicacid molecule of the invention. In one embodiment, the genetic constructis an expression construct. Also provided by the invention is a vectorcomprising the genetic construct, a host cell comprising the geneticconstruct or vector, a polypeptide encoded by a nucleic acid molecule ofthe invention, an antibody which selectively binds a polypeptide of theinvention, and a method for recombinantly producing a polypeptide of theinvention.

Accordingly, in another aspect the invention provides an isolated INS-SPbiomarker polypeptide or variant or fragment thereof selected from

-   -   (a) INS-SP (1-9) SEQ ID NO:16 or a variant or fragment thereof;    -   (b) INS-SP (15-24) SEQ ID NO:18 or a variant or fragment        thereof;    -   (c) an amino acid sequence having at least 70%, 75%, 80%, 85%,        90%, 95%, or 99% amino acid identity to (a) or (b), and    -   (d) an INS-SP polypeptide encoded by a nucleic acid molecule of        the invention.

The invention also relates to a use of a polypeptide of the invention inthe preparation of an anti-INS-SP biomarker antibody.

One method for recombinantly producing a polypeptide of the inventioncomprises the steps of:

-   -   (a) culturing a host cell comprising a genetic construct of the        invention capable of expressing a polypeptide of the invention;    -   (b) selecting cells expressing the polypeptide of the invention;    -   (c) separating the expressed polypeptide from the cells; and        optionally    -   (d) purifying the expressed polypeptide.

In one embodiment, the method comprises a pre-step of transfecting thehost cells with the construct.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described with reference to the figures in theaccompanying drawings in which

FIGURES

FIG. 1 is a bar graph showing circulating INS-SP biomarkerconcentrations in patients are derived from a cardiac source.

FIG. 2 shows the results of a radioimmunoassay showing concentrations ofINS-SP biomarker (filled circles) in plasma drawn from AMI patients(n=9) at the times shown from hospital admission. Highest levels ofINS-SP biomarker were seen at admission, being some 5 to 15 times higheron average than levels measured in normal healthy individuals;

FIG. 3 shows the results of a radioimmunoassay demonstratingimmunoreactive plasma Insulin-SPn biomarker, in contrast with Insulinitself, is significantly reduced in normal healthy volunteers by oralingestion of 75 g glucose, a common test for insulin sensitivity andrelease under metabolic loading.

FIG. 4 is a schematic diagram outlining the processing of humanpreprolnsulin resulting in the generation of free signal, C-peptide andinsulin α and β chains.

FIG. 5 shows a consensus alignment for insulin signal peptides frommouse, cat, sheep, pig, human and rat.

DEFINITIONS

Acute Cardiac Disorder (ACD) includes but is not limited to: acutecoronary syndromes including acute myocardial infarction (AMI) withST-elevation on presenting ECG, unstable angina, and acute nonST-elevated myocardial infarction; cardiac ischemia; acute cardiacinjury; acute cardiac damage resulting from acute drug toxicity, acutecardiomyopathies, and cardiac transplant rejection. Full descriptive,definitions of these disorders are found in reference 1.

ACD/pulmonary disorder refers to a subject with an undiagnosed, orsuspected ACD or pulmonary disorder.

Acute coronary syndromes (ACS) encompasses a wide spectrum of cardiacischemia events including unstable angina, acute myocardial infarct withST-elevation on presenting electrocardiogram (ECG), and acute myocardialinfarction without ST-segment elevation on ECG.

The term “antibody” refers to an immunoglobulin molecule having aspecific structure that interacts (binds) specifically with a moleculecomprising the antigen used for synthesizing the antibody or with anantigen closely related to it. As used herein, the term “antibody”broadly includes full length antibodies and may also include certainantibody fragments thereof. Also included are monoclonal and polyclonalantibodies, multivalent and monovalent antibodies, multispecificantibodies (for example bi-specific antibodies), chimeric antibodies,human antibodies, humanized antibodies and antibodies that have beenaffinity matured. An antibody binds selectively or specifically to anINS-SP polypeptide of the invention if the antibody binds preferentiallyto the INS-SP e.g. has less than 25%, or less than 10%, or less than 1%or less than 0.1% cross-reactivity with a non-INS-SP polypeptides.Usually, the antibody will have a binding affinity (dissociationconstant (Kd) value), for the antigen or epitope of no more than 10⁻⁶,or 10⁻⁷M, or less than about 10⁻⁸M, or 10⁻⁹M, or 10⁻¹⁰, or 10⁻¹¹ or10⁻¹²M. Binding affinity may be assessed using surface plasma resonance,for example, or standard Scatchard analysis.

As used herein, an “antigen-binding fragment” or “antibody fragment”means a portion of the intact antibody that retains the antigen bindingnormal of the antibody from which it was derived. Examples of antibodyfragments include Fab, Fab′, F(ab′)₂ and Fv fragments, linearantibodies, diabodies, single chain antibodies (ScFV) and multispecificantibodies.

As used herein, a “monoclonal antibody” means an antibody that is ahighly specific antibody directed against a single target antigen. Amonoclonal antibody may be obtained from a population of homogenous orsubstantially homogenous antibodies wherein each monoclonal antibody isidentical and/or bind the same epitope, except for natural mutationswhich may occur in minor amounts.

An “isolated antibody” is an identified antibody which has beenseparated or recovered, or both, from a component of its naturalenvironment. For example, separated from proteins including enzymes andhormones. In one embodiment, the antibody is purified to at least 95%,or 96% or 97% or 98% or 99% by weight of antibody. Purity can bedetermined by the Lowry method for example. Ordinarily the antibody willbe prepared by at least one purification step.

The term “binding agent” as used herein refers to any solid or non-solidmaterial capable of binding INS-SP or a fragment or variant thereof. Inone embodiment the term refers to any natural or non-natural moleculethat binds to INS-SP or a fragment or variant thereof. Examples ofbinding agents include proteins, peptides, nucleic acids, carbohydrates,lipids, and small molecule compounds. A selective or specific bindingagent is an antibody or antigen-binding fragment thereof.

Sample or biological sample as used herein means any sample taken orderived from a subject to be screened. The sample may be any sampleknown in the art in which the INS-SP biomarker can be detected. Includedare any body fluids such as plasma, blood, saliva, interstitial fluid,serum, urine, synovial, cerebrospinal, lymph, seminal, amniotic,pericardial fluid and ascites, as well as tissues such as cardiactissues but not limited thereto.

The term “epitope” includes any protein determinant capable of specificbinding to an immunoglobulin and/or T cell receptor. That is, a site onan antigen to which B and/or T cells respond. Epitopic determinantsusually consist of chemically active surface groupings of molecules suchas amino acids or sugar side chains, and usually have specific threedimensional structural characteristics, and specific chargecharacteristics. An epitope typically includes at least 3, 5 or usually8-10 amino acids. The amino acids may be contiguous, or non-contiguousamino acids juxtaposed by tertiary folding.

The term “within six hours of onset or clinical presentation includesfrom 1 minute up to and including 360 minutes from onset of, orpresentation at a medical facility for example with ACD, cardiactransplant rejection or an undiagnosed or suspected ACD/pulmonarydisorder. Measurements may be made within 4 hours (from 1 minute up toand including 240 minutes), within 2 hours (from 1 minute up to andincluding 120 minutes) or within 1 hour (from 1 minute up to andincluding 60 minutes) from onset or presentation, within 5 to 45minutes, 15 to 40 minutes, 20 to 35 minutes, or within 25 to 30 minutesof onset or presentation.

A level “higher” or “lower” than a control or reference value, or achange, difference, or deviation from a control or reference value inone embodiment is statistically significant. A higher level, lowerlevel, difference, or deviation from, or change from a control orreference level or mean control or reference level can be considered toexist if the level differs from the control or reference level by about5% or more, by about 10% or more, by about 20% or more, or by about 50%or more compared to the control or reference level. Statisticallysignificant may alternatively be calculated as P≤0.05. In a furtheralternative, higher levels, lower levels, deviation, and changes can bedetermined by recourse to assay reference limits or reference intervals.These can be calculated from intuitive assessment or non-parametricmethods. Overall, these methods calculate the 0.025, and 0.975 fractilesas 0.025*(n+1) and 0.975 (n+1). Such methods are well known in theart.^(22,23) Presence of a marker (including INS-SP) absent in acontrol, for example, is also contemplated as a higher level, deviationor change. Absence of a marker (including INS-SP) present in a controlis also contemplated as a lower level, deviation or change.

Included are samples taken or derived from any subjects such as fromnormal healthy subjects with no clinical history of biological events ordisorders, including glucose handling disorders, diabetes or ACD andsubjects with various ACDs including but not limited to acute coronarysyndromes: (AMI) with ST-elevation on presenting ECG, unstable angina,and acute non ST-elevated MI; cardiac ischemia; acute cardiac injury;acute cardiac damage resulting from acute drug toxicity, acutecardiomyopathies, and cardiac transplant rejection.

The term “cardiomyopathies” as used herein refers to diseases of themyocardium where the myocardium or heart muscle is weakened. This canresult in reduced pumping of the heart. Common causes ofcardiomyopathies are heart attacks, viral infections, high bloodpressure, alcoholism, and autoimmune diseases.

A biological event or disorder as used herein refers to a range ofevents in which an INS-SP biomarker is released into the circulation ofa subject, including both acute and chronic conditions. Examplarconditions include metabolic disorders such as obesity, diabetes, kidneydisease, a glucose handling disorder including metabolic syndrome,glucose intolerance, hyperglycemia, and insulin resistance;non-alcoholic fatty liver disease (including non-alcoholicsteatohepatitis) and fatty liver disease (including alcoholic liverdisease), cardiovascular disease (including ACD's such as but notlimited to acute coronary syndrome). Examples of chronic conditions arediabetes and cardiovascular disease.

The term INS-SP refers to the complete 24 amino acid INS signal peptidefor the human preproinsulin sequence (1-110)(SEQ ID NO: 1). INS-SP(1-24) is shown separately in SEQ ID NO:14. INS-SP biomarkers includeINS-SP, as well as INS-SP-derived or INS-SP-related polypeptidescomprising, consisting essentially of, or consisting of a variant orfragment of INS-SP. Fragments useful as INS-SP biomarkers include INS-SP(1-9) SEQ ID NO:16 and INS-SP (15-24) SEQ ID NO:18. In one embodimentINS-SP functions as a signal polypeptide, or as an antigenic polypeptideto which an antibody can bind. Variants and fragments of INS-SP includevariants and fragments which retain at least the antigenic function.

The term “comprising” as used in this specification and claims means“consisting at least in part of”; that is to say when interpretingstatements in this specification and claims which include “comprising”,the features prefaced by this term in each statement all need to bepresent but other features can also be present. Related terms such as“comprise” and “comprised” are to be interpreted in similar manner.

The term “diabetes” as used herein encompasses both Type 1 (diabetesmellitus) and Type 2 diabetes. Type 1 diabetes is defined as a state ofchronic hyperglycaemia. A venous plasma fasting glucose level of morethan 7.0 mmol/L and/or a value exceeding 11.1 mmol/L either 2 hoursafter a glucose tolerance test, or in a random sample is indicative oftype 1 diabetes (see Oxford Textbook of Medicine, Warrell et al; 4^(th)Ed, 2005, p 317).

The term “glucose handling disorder” as used herein includes variousstates of hyper- and hypoglycaemia (including metabolic syndrome).Hyperglycaemic states include impaired glucose tolerance (IGT) andimpaired fasting glucose (IFG). A venous plasma fasting glucose level ofless than 7.0 mmol/L and glucose tolerance test value at 2 hours ofbetween 7.8 and 11.1 mmol/L is indicative of IGT. Fasting glucose levelsof 6.1 to 6.9 mmol/L is indicative of IFG (see Oxford Textbook ofMedicine, Supra).

The term “glucose tolerance test” as used herein refers to the wellknown glucose test which commonly is administered after fasting by asubject drinking 75 g of anhydrous glucose dissolved in 250 ml of water(see Oxford Textbook of Medicine, Supra).

The term “polynucleotide(s),” as used herein, means a single ordouble-stranded deoxyribonucleotide or ribonucleotide polymer of anylength, and include as non-limiting examples, coding and non-codingsequences of a gene, sense and antisense sequences, exons, introns,genomic DNA, cDNA, pre-mRNA, mRNA, rRNA, siRNA, miRNA, tRNA, ribozymes,recombinant polynucleotides, isolated and purified naturally occurringDNA or RNA sequences, synthetic RNA and DNA sequences, nucleic acidprobes, primers, fragments, genetic constructs, vectors and modifiedpolynucleotides. Reference to a nucleic acid molecule is to be similarlyunderstood.

A “fragment” of a polynucleotide sequence provided herein is asubsequence of contiguous nucleotides that is capable of specifichybridization to a target of interest, e.g., a sequence that is at least10 nucleotides in length. In one embodiment the fragments of theinvention comprise at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, or 71, contiguousnucleotides of a polynucleotide of SEQ ID NO:15. A fragment of apolynucleotide sequence can be used as a primer, a probe, included in amicroarray, or used in polynucleotide-based selection methods herein.Fragments of other polynucleotides of the invention (such as SEQ IDNO:17 or SEQ ID NO:19) or polynucleotides described herein should besimilarly understood. For example, INS-SP (1-9) SEQ ID NO:17 and INS-SP(15-24) SEQ ID NO:19 fragments have at least 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 contiguousnucleotides of SEQ ID NO:17 or SEQ ID NO:19 respectively.

The term “primer” refers to a short polynucleotide, usually having afree 3′OH group, that is hybridized to a template and used for primingpolymerization of a polynucleotide complementary to the target.

The term “probe” refers to a short polynucleotide that is used to detecta polynucleotide sequence, that is complementary to the probe, in ahybridization-based assay. The probe may consist of a “fragment” of apolynucleotide as defined herein.

The term “polypeptide”, as used herein, encompasses amino acid chains ofany length, including full length sequences in which amino acid residuesare linked by covalent bonds. Polypeptides useful in the presentinvention may be purified natural products, or may be produced partiallyor wholly using recombinant or synthetic techniques. The term may referto a polypeptide, an aggregate of a polypeptide such as a dimer or othermultimer, a fusion polypeptide, a polypeptide fragment, a polypeptidevariant, or derivative thereof. Polypeptides herein may have chainlengths of at least 4 amino acids, at least 5 amino acids, at least 6,at least 7, at least 8, at least 9, at least 10, at least 11, at least12, at least 13, at least 14, at least 15, at least 16, at least 17, atleast 18, at least 19, at least 20, at least 21, at least 22, at least23, or all 24 amino acids of the full-length INS-SP protein (SEQ IDNO:14). Reference to other polypeptides of the invention (such as SEQ IDNO:16 or SEQ ID NO: 18), or other polypeptides described herein shouldbe similarly understood.

A “fragment” of a polypeptide is a subsequence of the polypeptide thatperforms a function that is required for the biological activity orbinding and/or provides three dimensional structure of the polypeptide.The term may refer to a polypeptide, an aggregate of a polypeptide suchas a dimer or other multimer, a fusion polypeptide, a polypeptidefragment, a polypeptide variant, or derivative thereof. In oneembodiment the fragment is capable of performing the above signalpeptide activity, or retains the antigenic-binding properties of INS-SP(1-24), INS-SP (1-9), or INS-SP (15-24), or other polypeptide of theinvention or polypeptide described herein.

The term “isolated” as applied to the polynucleotide or polypeptidesequences disclosed herein is used to refer to sequences that areremoved from their natural cellular environment. An isolated moleculemay be obtained by any method or combination of methods includingbiochemical, recombinant, and synthetic techniques. The polynucleotideor polypeptide sequences may be prepared by at least one purificationstep.

The term “purified” as used herein does not require absolute purity.Purified refers in one embodiment to at least 90%, or 95%, or 98%, or99% homogeneity of a polynucleotide, polypeptide antibody, or host cellin a sample. The term should be similarly understood in relation toother molecules and constructs described herein.

The term “isolated” as applied to a cell or host cell describes a cellor host cell that has been obtained or removed from an organism or fromits natural environment and is subsequently maintained in a laboratoryenvironment as known in the art. The term is not limited to singlecells, per se, but refers to a cell or host cell comprised in a cellculture and can include a single cell or single host cell.

The term “recombinant” refers to a polynucleotide sequence that isremoved from sequences that surround it in its natural context and/or isrecombined with sequences that are not present in its natural context.

A “recombinant” polypeptide sequence is produced by translation from a“recombinant” polynucleotide sequence.

As used herein, the term “variant” refers to polynucleotide orpolypeptide sequences different from the specifically identifiedsequences, wherein one to 18 or more nucleotides, and 1 to 6 or moreamino acid residues are deleted, substituted, or added. Substitutions of1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18nucleotides are specifically contemplated. Substitutions, additions ordeletions of one, two, three, four, five or six amino acids are alsocontemplated. Variants may be naturally occurring allelic variants, ornon-naturally occurring variants. Variants may be from the same or fromother species and may encompass homologues, paralogues and orthologues.In certain embodiments, variants of the polypeptides useful in theinvention have biological activities including signal peptide activityor antigenic-binding properties that are the same or similar to those ofthe parent polypeptides or polynucleotides. The term “variant” withreference to polynucleotides and polypeptides encompasses all forms ofpolynucleotides and polypeptides as defined herein.

Variant polynucleotide sequences exhibit at least 50%, at least 60%, atleast 70%, at least 71%, at least 72%, at least 73%, at least 74%, atleast 75%, at least 76%, at least 77%, at least 78%, at least 79%, atleast 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to a sequence of the present invention. Identity is found overa comparison window of at least 10 nucleotide positions, at least 15nucleotide positions, at least 20 nucleotide positions, at least 27nucleotide positions, at least 40 nucleotide positions, at least 50nucleotide positions, at least 60, at least 65, or at least 70nucleotide positions or over the entire length of a polynucleotide ofSEQ ID NO:15. For other polynucleotides disclosed herein. Identity maybe similarly determined. For example, for SEQ ID NO:17 or SEQ ID NO:19the comparison window may be at least 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotidepositions.

Polynucleotide sequence identity may be calculated over the entirelength of the overlap between a candidate and subject polynucleotidesequences using global sequence alignment programs (e.g. Needleman, S.B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). A fullimplementation of the Needleman-Wunsch global alignment algorithm isfound in the needle program in the EMBOSS package (Rice, P. Longden, I.and Bleasby, A. EMBOSS: The European Molecular Biology Open SoftwareSuite, Trends in Genetics June 2000, vol 16, No 6. pp. 276-277) whichcan be obtained from http://www.hgmp.mrc.ac.uk/Software/EMBOSS/. TheEuropean Bioinformatics Institute server also provides the facility toperform EMBOSS-needle global alignments between two sequences on line athttp:/www.ebi.ac.uk/emboss/align/.

Alternatively the GAP program may be used which computes an optimalglobal alignment of two sequences without penalizing terminal gaps. GAPis described in the following paper: Huang, X. (1994) On Global SequenceAlignment (Computer Applications in the Biosciences 10, 227-235).

Polynucleotide variants also encompass those which exhibit a similarityto one or more of the specifically identified sequences that is likelyto preserve the functional equivalence of those sequences and whichcould not reasonably be expected to have occurred by random chance.

This program finds regions of similarity between the sequences and foreach such region reports an “E value” which is the expected number oftimes one could expect to see such a match by chance in a database of afixed reference size containing random sequences. The size of thisdatabase is set by default in the bl2seq program. For small E values,much less than one, the E value is approximately the probability of sucha random match.

Variant polynucleotide sequences preferably exhibit an E value of lessthan 1×10⁻⁵, less than 1×10⁻⁶, less than 1×10⁻⁹, less than 1×10⁻¹², lessthan 1×10⁻¹⁵, less than 1×10⁻¹⁸ or less than 1×10⁻²¹ when compared withany one of the specifically identified sequences.

Polynucleotide sequence identity and similarity can also be determinedin the following manner. The subject polynucleotide sequence is comparedto a candidate polynucleotide sequence using sequence alignmentalgorithms and sequence similarity search tools such as in Genbank,EMBL, Swiss-PROT and other databases. Nucleic Acids Res 29:1-10 and11-16, 2001 provides examples of online resources.

Use of BLASTN is preferred for use in the determination of sequenceidentity for polynucleotide variants according to the present invention.

BLASTN (from the BLAST suite of programs, version 2.2.18 April 2008 inbl2seq (Tatiana A. et al, FEMS Microbiol Lett. 174:247-250 (1999),Altschul et al., Nuc. Acis Res 25:3389-3402, (1997)), is publiclyavailable from NCBI (ftp://ftp.ncbi.nih.gov/blast/) or from NCB1 atBethesda, Md., USA. The default parameters of bl2seq are utilized exceptthat filtering of low complexity parts should be turned off.

The identity of polynucleotide sequences may be examined using thefollowing UNIX command line parameters:

bl2seq −i nucleotideseq1 −j nucleotideseq2 −p blastn

The parameter −F F turns off filtering of low complexity sections. Theparameter −p selects the appropriate algorithm for the pair ofsequences. The bl2seq program reports sequence identity as both thenumber and percentage of identical nucleotides in a line “Identities=”.

Alternatively, variant polynucleotides are polynucleotides thathybridize to the specified polynucleotide sequence, or a complementthereof under stringent conditions.

The term “hybridize under stringent conditions”, and grammaticalequivalents thereof, refers to the ability of a polynucleotide moleculeto hybridize to a target polynucleotide molecule (such as a targetpolynucleotide molecule immobilized on a DNA or RNA blot, such as aSouthern blot or Northern blot) under defined conditions of temperatureand salt concentration. The ability to hybridize under stringenthybridization conditions can be determined by initially hybridizingunder less stringent conditions then increasing the stringency to thedesired stringency.

With respect to polynucleotide molecules greater than about 100 bases inlength, typical stringent hybridization conditions are no more than 25to 30° C. (for example, 10° C.) below the melting temperature (Tm) ofthe native duplex (see generally, Sambrook et al., Eds, 1987, MolecularCloning, A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press; Ausubelet al., 1987, Current Protocols in Molecular Biology, Greene Publishing,incorporated herein by reference). Tm for polynucleotide moleculesgreater than about 100 bases can be calculated by the formula Tm=81.5+0.41% (G+C-log (Na+) (Sambrook et al., Eds, 1987, Molecular Cloning, ALaboratory Manual, 2nd Ed. Cold Spring Harbor Press; Bolton andMcCarthy, 1962, PNAS 84:1390). Typical stringent conditions for apolynucleotide of greater than 100 bases in length would behybridization conditions such as prewashing in a solution of 6×SSC, 0.2%SDS; hybridizing at 65° C., 6×SSC, 0.2% SDS overnight; followed by twowashes of 30 minutes each in 1×SSC, 0.1% SDS at 65° C. and two washes of30 minutes each in 0.2×SSC, 0.1% SDS at 65° C.

In one embodiment stringent conditions use 50% formamide, 5×SSC, 50 mMsodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt'ssolution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10%dextran sulphate at 42° C., with washes at 42° C. in 0.2×SSC and 50%formamide at 55° C., followed by a wash comprising of 0.1×SSC containingEDTA at 55° C.

With respect to polynucleotide molecules having a length less than 100bases, exemplary stringent hybridization conditions are 5 to 10° C.below Tm. On average, the Tm of a polynucleotide molecule of length lessthan 100 bp is reduced by approximately (500/oligonucleotide length)° C.

With respect to the DNA mimics known as peptide nucleic acids (PNAs)(Nielsen et al., Science. 1991 Dec. 6; 254(5037):1497-500) Tm values arehigher than those for DNA-DNA or DNA-RNA hybrids, and can be calculatedusing the formula described in Giesen et al., Nucleic Acids Res. 1998Nov. 1; 26(21):5004-6. Exemplary stringent hybridization conditions fora DNA-PNA hybrid having a length less than 100 bases are 5 to 10° C.below the Tm.

Variant polynucleotides also encompasses polynucleotides that differfrom the sequences of the invention but that, as a consequence of thedegeneracy of the genetic code, encode a polypeptide having similaractivity to a polypeptide encoded by a polynucleotide of the presentinvention. A sequence alteration that does not change the amino acidsequence of the polypeptide is a “silent variation”. Except for ATG(methionine) and TGG (tryptophan), other codons for the same amino acidmay be changed by art recognized techniques, e.g., to optimize codonexpression in a particular host organism.

Polynucleotide sequence alterations resulting in conservativesubstitutions of one or several amino acids in the encoded polypeptidesequence without significantly altering its biological activity are alsoincluded in the invention. A skilled artisan will be aware of methodsfor making phenotypically silent amino acid substitutions (see, e.g.,Bowie et al., 1990, Science 247, 1306).

Variant polynucleotides due to silent variations and conservativesubstitutions in the encoded polypeptide sequence may be determinedusing the bl2seq program via the tblastx algorithm as described above.

The term “variant” with reference to polypeptides also encompassesnaturally occurring, recombinantly and synthetically producedpolypeptides. Variant polypeptide sequences preferably exhibit at least50%, at least 60%, at least 70%, at least 71%, at least 72%, at least73%, at least 74%, at least 75%, at least 76%, at least 77%, at least78%, at least 79%, at least 80%, at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% identity to a sequence of the present invention.Identity is found over a comparison window of at least 5, at least 7, atleast 10, at least 15, at least 20, at least 21, at least 22, at least23, amino acid positions, or over the entire length of a polypeptide ofSEQ ID NO:14, or other polypeptides disclosed or used in the invention.For example, for SEQ ID NO:16 or SEQ ID NO:18 the comparison window maybe at least 5, 6, 7, 8 or 9 amino acid positions, or over the entirelength of the polypeptide.

Polypeptide variants also encompass those which exhibit a similarity toone or more of the specifically identified sequences that is likely topreserve the functional equivalence of those sequences and which couldnot reasonably be expected to have occurred by random chance. Asdiscussed above, in the case of INS-SP variants function may be aseither a signal polypeptide, or antigenic polypeptide, or both.

Polypeptide sequence identity and similarity can be determined in thefollowing manner. The subject polypeptide sequence is compared to acandidate polypeptide sequence using BLASTP (from the BLAST suite ofprograms, version 2.2.18 [April 2008]) in bl2seq, which is publiclyavailable from NCBI (ftp://ftp.ncbi.nih.gov/blast/). The defaultparameters of bl2seq are utilized except that filtering of lowcomplexity regions should be turned off.

The similarity of polypeptide sequences may be examined using thefollowing UNIX command line parameters:

-   -   bl2seq −i peptideseq1 −j peptideseq2 −F F −p blastp

The parameter −F F turns off filtering of low complexity sections. Theparameter −p selects the appropriate algorithm for the pair ofsequences. This program finds regions of similarity between thesequences and for each such region reports an “E value” which is theexpected number of times one could expect to see such a match by chancein a database of a fixed reference size containing random sequences. Forsmall E values, much less than one, this is approximately theprobability of such a random match.

Variant polypeptide sequences commonly exhibit an E value of less than1×10⁻⁵, less than 1×10⁻⁶, less than 1×10⁻⁹, less than 1×10⁻¹², less than1×10⁻¹⁵, less than 1×10⁻¹⁸ or less than 1×10⁻²¹ when compared with anyone of the specifically identified sequences.

Polypeptide sequence identity may also be calculated over the entirelength of the overlap between a candidate and subject polypeptidesequences using global sequence alignment programs. EMBOSS-needle(available at http:/www.ebi.ac.uk/emboss/align/) and GAP (Huang, X.(1994) On Global Sequence Alignment. Computer Applications in theBiosciences 10, 227-235.) as discussed above are also suitable globalsequence alignment programs for calculating polypeptide sequenceidentity.

Use of BLASTP as described above is preferred for use in thedetermination of polypeptide variants according to the presentinvention.

In one embodiment variants include peptides who's sequence differs fromthe human INS-SP (1-24) SEQ ID NO:14, INS-SP (1-9) SEQ ID NO:16 orINS-SP (15-24) SEQ ID NO:18 herein by one, two, three, four, five, sixor more conservative or non-conservative amino acid substitutions,deletions or additions or insertions conservative mutations do notaffect the biological activity of the peptide. Conservativesubstitutions typically include the substitution of one amino acid foranother with similar characteristics, e.g., substitutions within thefollowing groups: valine, glycine; glycine, alanine; valine, isoleucine,leucine; aspartic acid, glutamic acid; asparagines, glutamine; serine,threonine; lysine, arginine; and phenylalanine, tyrosine. Examples ofconservative substations can also be found in the sequences of INS-SP asshown in the sequence listings whereby the substitutions in differentmammalian species compared to the human sequence are shown. Otherconservative substitutions can be taken from FIG. 5 and Table 1 below.

TABLE 1 Original Exemplary Conservative Other Residue Substitutionssubstitutions Ala (A) val; leu; ile ser, thr, gln, his, arg Arg (R) lys;gln; asn his Asn (N) gln; his; lys; arg Asp (D) glu lys, ala Cys (C) serGln (Q) asn Glu (E) asp Gly (G) pro; ala ile, glu His (H) asn; gln; lys;arg Ile (I) leu; val; met; ala; phe; norleucine Leu (L) norleucine; ile;val; met; ala; phe pro Lys (K) arg; gln; asn Met (M) leu; phe; ile thr,val Phe (F) leu; val; ile; ala; tyr Pro (P) ala Ser (S) thr Thr (T) serTrp (W) tyr; phe leu Tyr (Y) trp; phe; thr; ser Val (V) ile; leu; met;phe; ala; norleucine

Naturally occurring residues are divided into groups based on commonside-chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gln, his, lys, arg:

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for a member of another class.

Other variants include peptides with modifications which influencepeptide stability. Such analogs may contain, for example, one or morenon-peptide bonds (which replace the peptide bonds) in the peptidesequence. Also included are analogs that include residues other thannaturally occurring L-amino acids, e.g. D-amino acids or non-naturallyoccurring synthetic amino acids, e.g. beta or gamma amino acids andcyclic analogs.

Substitutions, deletions, additions or insertions may be made bymutagenesis methods known in the art. A skilled worker will be aware ofmethods for making phenotypically silent amino acid substitutions. Seefor example Bowie et al., 1990, Science 247, 1306.⁹, Kunkel, T; 1985,PNAS, 85 p 488.²⁷

Also included within the polypeptides of the invention are those whichhave been modified during or after synthesis for example bybiotinylation, benzylation, glycosylation, phosphorylation, amidation,by derivatization using blocking/protecting groups and the like. Suchmodifications may increase stability or activity of the polypeptide.Such modifications are well known in the art. See for example, Sambrookand Ausubel (supra), and Lundblad, R, CRC Press, 1995.²⁸

The term “genetic construct” refers to a polynucleotide molecule,usually double-stranded DNA, which may have inserted into it anotherpolynucleotide molecule (the insert polynucleotide molecule) such as,but not limited to, a cDNA molecule. A genetic construct may contain thenecessary elements that permit transcribing the insert polynucleotidemolecule, and, optionally, translating the transcript into apolypeptide. The insert polynucleotide molecule may be derived from thehost cell, or may be derived from a different cell or organism and/ormay be a recombinant polynucleotide. Once inside the host cell thegenetic construct may become integrated in the host chromosomal DNA. Thegenetic construct may be linked to a vector.

The term “vector” refers to a polynucleotide molecule, usually doublestranded DNA, which is used to transport the genetic construct into ahost cell. The vector may be capable of replication in at least oneadditional host system, such as E. coli.

The term “expression construct” refers to a genetic construct thatincludes the necessary elements that permit transcribing the insertpolynucleotide molecule, and, optionally, translating the transcriptinto, a polypeptide. An expression construct typically comprises in a 5′to 3′ direction:

-   -   (a) a promoter functional in the host cell into which the        construct will be transformed,    -   (b) the polynucleotide to be expressed, and    -   (c) a terminator functional in the host cell into which the        construct will be transformed.

The term “coding region” or “open reading frame” (ORF) refers to thesense strand of a genomic DNA sequence or a cDNA sequence that iscapable of producing a transcription product and/or a polypeptide underthe control of appropriate regulatory sequences. The coding sequence isidentified by the presence of a 5′ translation start codon and a 3′translation stop codon. When inserted into a genetic construct, a“coding sequence” is capable of being expressed when it is operablylinked to promoter and terminator sequences and/or other regulatoryelements.

“Regulatory elements” and “polynucleotide regulatory elements” mean anyelement that controls or influences the expression of a polynucleotideinsert from a vector, genetic construct or expression cassette andincludes promoters, transcription control sequences, translation controlsequences, origins of replication, tissue-specific regulatory elements,temporal regulatory elements, enhancers, polyadenylation signals,repressors and terminators. Regulatory elements can be homologous orheterologous to the polynucleotide insert to be expressed from a vector,genetic, construct or expression cassette according to the invention.

“Homologous” as used herein with reference to the relationship between apolynucleotide regulatory element (PRE) and the sequence to which thePRE is operably linked in a genetic construct means that the PRE isnormally associated in nature with the coding sequence to which it isoperably linked in the construct. A homologous polynucleotide regulatoryelement may be operably linked to a polynucleotide of interest such thatthe polynucleotide of interest can be expressed from a, vector, geneticconstruct or expression cassette according to the invention.

“Heterologous” as used herein with reference to the relationship betweena polynucleotide regulatory element (PRE) and the sequence to which thePRE is operably linked in a genetic construct means that the PRE is notnormally associated in nature with the coding sequence to which it isoperably linked in the construct. Such PREs may include promotersnormally associated with different genes (other than INS), and/orpromoters isolated from any other bacterial, viral, eukaryotic, ormammalian cell.

“Operably-linked” means that the sequence to be expressed is placedunder the control of regulatory elements that include promoters,transcription control sequences, translation control sequences, originsof replication, tissue-specific regulatory elements, temporal regulatoryelements, enhancers, polyadenylation signals, repressors andterminators.

The term “noncoding region” refers to untranslated sequences that areupstream of the translational start site and downstream of thetranslational stop site. These sequences are also referred torespectively as the 5′ UTR and the 3′ UTR. These regions includeelements required for transcription initiation and termination and forregulation of translation efficiency.

Terminators are sequences, which terminate transcription, and are foundin the 3′ untranslated ends of genes downstream of the translatedsequence. Terminators are important determinants of mRNA stability andin some cases have been found to have spatial regulatory functions.

The term “promoter” refers to nontranscribed cis-regulatory elementsupstream of the coding region that regulate gene transcription.Promoters comprise cis-initiator elements which specify thetranscription initiation site and conserved boxes such as the TATA box,and motifs that are bound by transcription factors.

The terms “to alter expression of” and “altered expression” of apolynucleotide or polypeptide of the invention, are intended toencompass the situation where genomic DNA corresponding to apolynucleotide of the invention is modified thus leading to alteredexpression of a polynucleotide or polypeptide of the invention.Modification of the genomic DNA may be through genetic transformation orother methods known in the art for inducing mutations. The “alteredexpression” can be related to an increase or decrease in the amount ofmessenger RNA and/or polypeptide produced and may also result in alteredactivity of a polypeptide due to alterations in the sequence of apolynucleotide and polypeptide produced.

“Subject” as used herein is preferably a mammal and includes human, andnon-human mammals such as cats, dogs, horses, cows, sheep, deer, mice,rats, primates (including gorillas, rhesus monkeys and chimpanzees),possums and other domestic farm or zoo animals. In one embodiment, themammal is human.

The term “presentation” as used herein refers to presentation of asubject at a medical facility such as a clinic or hospital.

A “therapeutically effective amount” or “therapeutically effective dose”as used herein means an amount sufficient to produce the desiredphysiological effect or an amount capable of achieving the desiredresult, particularly for treating the desired disease or condition,including reducing or eliminating one or more symptoms or manifestationsof the disease or condition.

The term “treat”, “treating” or “treatment” and “preventing” refer totherapeutic or prophylactic measures which alleviate, ameliorate,manage, prevent, restrain, stop or reverse progression of a biologicalevent characterized by an INS-SP level which shows a deviation fromnormal control levels, including a glucose handling disorder, diabetes,hyperglycemia, obesity, ACD, or cardiac transplant rejection or effectsthereof, particularly of ACS. The subject may show observable ormeasurable (statistically significant) reduction in one or more ofglucose, lactate, insulin, fatty acids, triglycerides, Tn, TnI, TnT BNP,N-BNP, BNP-SP, BNP-SP fragments, ANP, ANP-SP, ANP-SP fragments, creatinekinase-MB, myoglobin, LDH, aspartate aminotransferase, H-FABP,endothelin, adrenomedullin, ischemia modified albumin, renin,angiotensin II, and other usual clinical markers known to those skilledin the art, indicating improvement.

The term “mass spectrometry” as used herein refers to methods offiltering, detecting, and measuring ions based on their mass to chargeratio. See for example U.S. Pat. No. 5,719,060, U.S. Pat. No. 6,204,500,U.S. Pat. No. 6,107,623, U.S. Pat. No. 6,124,137, U.S. Pat. No.6,225,047, U.S. Pat. No. 6,268,144, U.S. Pat. No. 7,057,165, and U.S.Pat. No. 7,045,366. Common mass spectrometry techniques includematrix-assisted laser desorption ionization (MALDI) and surface-enhancedlaser desorption ionization (SELDI). Both may be coupled with time offlight analysers (MALDI-TOF and SELDI-TOF) which allow for analysis ofanalytes at femtomole levels in very short ion pulses.

Versions of SELDI discussed for example in U.S. Pat. No. 5,719,600, U.S.Pat. No. 6,124,137, and U.S. Pat. No. 6,225,047 which are useful in thisinvention include Surface-Enhanced Affinity Capture (SEAC),Surface-Enhanced Neat Desorption (SEND), and Surface-EnhancedPhotolabile Attachment and Release (SEPAR).

It is intended that reference to a range of numbers disclosed herein(for example 1 to 10) also incorporates reference to all related numberswithin that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9and 10) and also any range of rational numbers within that range (forexample 2 to 8, 1.5 to 5.5 and 3.1 to 4.7) and, therefore, allsub-ranges of all ranges expressly disclosed herein are expresslydisclosed. These are only examples of what is specifically intended andall possible combinations of numerical values between the lowest valueand the highest value enumerated are to be considered to be expresslystated in this application in a similar manner.

DETAILED DESCRIPTION OF THE INVENTION

Insulin (INS) is a well known polypeptide hormone secreted by the (3cells of the pancreas. It has extensive effects on metabolism. A primaryrole of insulin is to cause cells to take up glucose from blood and tostore it as glycogen in liver and muscle, and stop use of fat as anenergy source. In diabetes insulin levels are low or even absent,thereby detrimentally affecting glucose handling.

As shown in SEQ ID NO:1, preproinsulin is a 110 amino acid molecule. Itconsists of two polypeptide chains (A and B), linked by disulsphidebridges. Preproinsulin (1-110) is cleaved to give a signal peptide of 24amino acids (SEQ ID NO:14), C-peptide and insulin. Processing of humanpreproinsulin is shown in FIG. 5.

It has long been thought that the functional role of the INS-SP islimited to controlling the trafficking of insulin in the endoplasmicreticulum. Once this is achieved it has been assumed that the signalpeptide is then degraded without ever being secreted from the cell.²⁵

Confounding customary views, the present applicants have now found thatINS-SP, typically in the form of INS-SP fragments, appears in thecirculation. This finding itself means INS-SP and INS-SP fragments areuseful as a circulating biomarker for a range of biological events. Forexample, it is anticipated that in diabetics and undiagnosed diabetics,for example, the level of ISN-SP will be above or below the normalcontrol or reference level, depending on whether the subject is hypo- orhyper-insulinemic. A lower level is symptomatic of a deficiency ininsulin action or secretion.

Accordingly, in one aspect, the invention provides a method forpredicting, diagnosing or monitoring a biological event in a subjectwherein the event correlates with the release of an INS-SP biomarkerinto the circulation, the method comprising:

-   -   (a) measuring the level of INS-SP biomarker in a biological        sample from the subject; and    -   (b) comparing the level of INS-SP biomarker with the INS-SP        level from a control or reference value,    -   wherein a deviation in the measured level from the control or        reference level is indicative of a biological event or disorder.

The biological event or disorder includes glucose handling disorders,diabetes and ACD.

The invention therefore also provides a method for assessing glucosehandling in a subject, the method comprising:

-   -   (a) measuring the level of INS-SP biomarker in a subject after        administration of glucose; and    -   (b) comparing the level of said INS-SP biomarker with the INS-SP        from a control or reference,    -   wherein a deviation in the measured level of INS-SP from the        control or reference level is indicative of a glucose handling        disorder.

Commonly, the deviation will be a lower measured level of INS-SPcompared to a control level. For example, in subjects withhyperglycaemia.³¹

In this method, glucose may be administered as a first step, accordingto the well known glucose tolerance test protocol (Oxford Textbook ofMedicine, Supra).

Assessments of plasma concentrations of INS-SP biomarkers, usuallyvenous plasma INS-SP, may be made at 2 hours after the glucose test isadministered in accordance with standard protocols. However,intermediate measurements for example at 15, 30, 45, 60, 90 and 105minutes after administration of the glucose are also useful.

The invention also provides a method for predicting, diagnosing,assessing or monitoring diabetes, or diabetic potential in a subject,the method comprising:

-   -   (a) measuring the level of INS-SP biomarker in a biological        sample from the subject; and    -   (b) comparing the level of INS-SP biomarker with the INS-SP        level from a control or reference,    -   wherein a measured level of INS-SP biomarker higher or lower        than the control level is indicative of diabetes or a        predisposition to diabetes.³¹

Whether the INS-SP biomarker level is higher or lower than normal willdepend on the insulin state of the subject.

The applicants have also surprisingly found that in patients with acutemyocardial infarction (AMI) the circulating concentration of INS-SP ishighest in the first few hours following the onset of the patient'ssymptoms—in fact, at the time of presentation to the hospital or clinic.Levels observed in the first two to six hours, or four hours weresurprisingly very high often reaching a peak some five to fifteen foldhigher than levels in a normal control population. There has been noprevious suggestion of the use of insulin or INS-SP or INS-SP fragmentsas a marker for ACD, cardiac transplant rejection or for use onundiagnosed or suspected ACD or pulmonary disorders.

These findings suggest INS-SP biomarkers are useful as a very clearearly stage marker of cardiac transplant rejection, ACD including acutecoronary syndromes (ACS) such as AMI, particularly non-ST elevated MI,and acute cardiac ischemia, and may be used to distinguish ACD frompulmonary disorders.

Based on these surprising findings, the applicants have determined forthe first time, that it would be useful to screen for circulating INS-SPor variants or fragments thereof, as well as, or alternately nucleotidesequences encoding INS-SP or the variants and fragments thereof in abiological sample taken from a subject, particularly within about six,about four or about two hours of onset of, or at clinical presentationwith the disorder.

Useful in the invention are antigenic fragments or variants of INS-SPwhich are least 4 or 5 amino acids in length. Peptides having as few as4 amino acids are known to be biologically active. See for exampleGilchrist et al, Biology and Reproduction, 21, 732-739, 2004; and Selaet al., Behring Ins. Mitt., 91, 54-66, 1992. Particularly usefulfragments are at the N-terminus (1-10) or C-terminus (15-24) of INS-SP.Examples of specific antigenic peptides are INS-SP (1-9) SEQ ID NO:16and INS-SP (15-24) SEQ ID NO:18. Corresponding nucleotide sequences aregiven in SEQ ID NOs: 17 and 19 respectively. These sequences areprovided by the applicants for the first time. Both the nucleic acidmolecules and peptides form aspects of the invention.

Accordingly, in another aspect, the invention provides a nucleic acidmolecule encoding an INS-SP fragment wherein said nucleic acid is

-   -   (a) SEQ ID NO:17 or a variant or fragment thereof;    -   (b) SEQ ID NO:19 or a variant or fragment thereof;    -   (c) a sequence which has at least 70%, 75%, 80%, 90%, 95% or 99%        sequence identity to SEQ ID NO:17 or SEQ ID NO:19;    -   (d) a sequence of at least 10 nucleotides in length capable of        hybridising under stringent conditions to (a) or (b); or    -   (e) a complement of any one of (a) to (d);

with the proviso that the sequence is not SEQ ID NO:15. SEQ ID NO:15 isthe full length nucleic acid sequence encoding the signal peptide.

The invention also provides isolated INS-SP polypeptides and INS-SPfragment polypeptides encoded by a nucleic acid molecule of theinvention.

Specific polypeptides of the invention include polypeptides having theamino acid sequences of SEQ ID NOs: 16 and 18 all as set forth in theaccompanying sequence listing. Also contemplated are variants andfragments of these polypeptides as defined herein, or amino acidsequences having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% amino acididentity to the polypeptide of SEQ ID NO:16 or SEQ ID NO:18. In oneembodiment the variants or fragments are functionally equivalentvariants or fragments. That is the variants or fragments maintain thefunction of SEQ ID NO:16 or SEQ ID NO:18 as antigens or signal peptides.The known full length INS-SP (1-24) SEQ ID NO:14 is not claimed per se,but is useful in the present invention. For example, the polypeptidesmay be used in the preparation of anti-INS-SP antibodies.

The nucleic acid molecules of the invention or otherwise describedherein are in one embodiment isolated. They can be isolated from abiological sample using a variety of techniques known to those ofordinary skill in the art. By way of example, such polynucleotides canbe isolated through use of the polymerase chain reaction (PCR) describedin Mullis et al., Eds. 1994 The Polymerase Chain Reaction, Birkhauser.The nucleic acid molecules of the invention can be amplified usingprimers, as defined herein, derived from the polynucleotide sequences ofthe invention. (See for example Mullis, Sambrook supra; and MolecularDiagnostic. PCR Handbook Gerrit, V et al., Springer, 2005).

Further methods for isolating polynucleotides include use of all, orportions of, the polynucleotide of the invention, particularlypolynucleotides having the sequence set forth in SEQ ID NOs:17 or SEQ IDNO:19 as hybridization probes. The technique of hybridizing labeledpolynucleotide probes to polynucleotides immobilized on solid supportssuch as nitrocellulose filters or nylon membranes, can be used to screengenomic or cDNA libraries. Similarly, probes may be coupled to beads andhybridized to the target sequence. Isolation can be effected using knownart protocols such as magnetic separation. Exemplary stringenthybridization and wash conditions are as given above.

Polynucleotide fragments may be produced by techniques well-known in theart such as restriction endonuclease digestion and oligonucleotidesynthesis.

A partial polynucleotide sequence may be used as a probe, in methodswell-known in the art to identify the corresponding full lengthpolynucleotide sequence in a sample. Such methods include PCR-basedmethods, 5′RACE (Methods Enzymol. 218: 340-56 (1993); Sambrook et al.,Supra) and hybridization-based method, computer/database-based methods.Detectable labels such as radioisotopes, fluorescent, chemiluminescentand bioluminescent labels may be used to facilitate detection. InversePCR also permits acquisition of unknown sequences, flanking thepolynucleotide sequences disclosed herein, starting with primers basedon a known region (Triglia et al., Nucleic Acids Res 16, 8186, (1998))The method uses several restriction enzymes to generate a suitablefragment in the known region of a gene. The fragment is thencircularized by intramolecular ligation and used as a PCR template.Divergent primers are designed from the known region. In order tophysically assemble full-length clones, standard molecular biologyapproaches can be utilized (Sambrook et al., Supra). Primers and primerpairs which allow amplification of polynucleotides of the invention,also form a further aspect of this invention.

Variants (including orthologues) may be identified by the methodsdescribed. Variant polynucleotides may be identified using PCR-basedmethods (Mullis et al., Eds. 1994 The Polymerase Chain Reaction,Birkhauser). Typically, the polynucleotide sequence of a primer, usefulto amplify variants of polynucleotide molecules by PCR, may be based ona sequence encoding a conserved region of the corresponding amino acidsequence.

Further methods for identifying variant polynucleotides include use ofall, or portions of, the specified polynucleotides as hybridizationprobes to screen genomic or cDNA libraries as described above. Typicallyprobes based on a sequence encoding a conserved region of thecorresponding amino acid sequence may be used. Hybridisation conditionsmay also be less stringent than those used when screening for sequencesidentical to the probe.

The variant sequences, including both polynucleotide and polypeptidevariants, may also be identified by the computer-based methods discussedabove.

In addition, multiple sequence alignments of a group of relatedsequences can be carried out with CLUSTALW (Thompson, et al., NucleicAcids Research, 22:4673-4680 (1994),http://www-igbmc.u-strasbg.fr/BioInfo/ClustalW/Top.html) or T-COFFEE(Cedric Notredame et al., J. Mol.

Biol. 302: 205-217 (2000))) or PILEUP, which uses progressive, pairwisealignments. (Feng et al., J. Mol. Evol. 25, 351 (1987)).

Pattern recognition software applications are available for findingmotifs or signature sequences. For example, MEME (Multiple Em for MotifElicitation) finds motifs and signature sequences in a set of sequences,and MAST (Motif Alignment and Search Tool) uses these motifs to identifysimilar or the same motifs in query sequences. The MAST results areprovided as a series of alignments with appropriate statistical data anda visual overview of the motifs found. MEME and MAST were developed atthe University of California, San Diego.

PROSITE (Bairoch et al., Nucleic Acids Res. 22, 3583 (1994); Hofmann etal., Nucleic Acids Res. 27, 215 (1999)) is a method of identifying thefunctions of uncharacterized proteins translated from genomic or cDNAsequences. The PROSITE database (www.expasy.org/prosite) containsbiologically significant patterns and profiles and is designed so thatit can be used with appropriate computational tools to assign a newsequence to a known family of proteins or to determine which knowndomain(s) are present in the sequence (Falquet et al., Nucleic AcidsRes. 30, 235 (2002)). Prosearch is a tool that can search SWISS-PROT andEMBL databases with a given sequence pattern or signature.

Proteins can be classified according to their sequence relatedness toother proteins in the same genome (paralogues) or a different genome(orthologues). Orthologous genes are genes that evolved by speciationfrom a common ancestral gene and normally retain the same function asthey evolve. Paralogous genes are genes that are duplicated within agenome and genes may acquire new specificities or modified functionswhich may be related to the original one. Phylogenetic analysis methodsare reviewed in Tatusov et al., Science 278, 631-637, 1997.

As noted above, the invention also relates to INS-SP polypeptidesencoded by the nucleic acid molecules of the invention, and includesvariants and fragments of these polypeptides.

In addition to the computer/database methods described above,polypeptide variants may be identified by physical methods, for exampleby screening expression libraries using antibodies raised againstpolypeptides of the invention (Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987) byrecombinant DNA techniques also described by Sambrook et al. or byidentifying polypeptides from natural sources with the aid of suchantibodies.

Polypeptides, including variant polypeptides, may be prepared usingpeptide synthesis methods well known in the art such as direct peptidesynthesis using solid phase techniques (e.g. Merrifield, 1963, in J. AmChem. Soc. 85, 2149; Stewart et al., 1969, in Solid-Phase PeptideSynthesis, WH Freeman Co, San Francisco Calif.; Matteucci et al. J. Am.Chem. Soc. 103:3185-3191, 1981, and Atherton et al., in Solid PhasePeptide Synthesis: a practical approach. IRL press (1989)) or automatedsynthesis, for example using a Synthesiser from Applied Biosystems(California, USA). Mutated forms of the polypeptides may also beproduced using synthetic methods such as site-specific mutagensis of theDNA encoding the amino acid sequence as described by Adelmen et al; DNA2, 183(1983). See also Protein Protocols Handbook; Walker, J. HumanaPress 2002.

The polypeptides and variant polypeptides herein are in one embodimentisolated. They may be isolated or purified from natural sources using avariety of techniques that are well known in the art (e.g. Deutscher,1990, Ed, Methods in Enzymology, Vol. 182, Guide to ProteinPurification, and Protein Protocols Handbook, supra). Technologiesinclude HPLC, ion-exchange chromatography, and immunochromatography butare not limited thereto.

Alternatively the polypeptides and variant polypeptides may be expressedrecombinantly in suitable host cells and separated from the cells asdiscussed below. The polypeptides and variants have utility ingenerating antibodies, and generating ligands amongst other uses.

The genetic constructs described herein may comprise one or more of thedisclosed polynucleotide sequences and/or polynucleotides encoding thedisclosed polypeptides, of the invention and may be useful fortransforming, for example, bacterial, fungal, insect, mammalian or plantorganisms. The genetic constructs of the invention are intended toinclude expression constructs as herein defined. Included are vectors(such as pBR322, pUC18, pU19, Mp18, Mp19, ColE1, PCR1 and pKRC), phages(such as lambda gt10), and M13 plasmids (such as pBR322, pACYC184,pT127, RP4, p1J101, SV40 and BPV), cosmids, YACS, BACs shuttle vectorssuch as pSA3, PAT28 transposons (such as described in U.S. Pat. No.5,792,294) and the like.

The constructs may conveniently include a selection gene or selectablemarker. Typically an antibiotic resistance marker such as ampicillin,methotrexate, or tetracycline is used.

Promoters useful in the constructs include β-lactamase, alkalinephosphatase, tryptophan, and tac promoter systems which are all wellknown in the art. Yeast promoters include 3-phosphoglycerate kinase,enolase, hexokinase, pyruvate decarboxylase, glucokinase, andglyceraldehydrate-3-phosphanate dehydrogenase but are not limitedthereto.

Enhancers may also be employed to act on the promoters to enhancetranscription. Suitable enhancers for use herein include SV40 enhancer,cytomeglovirus early promoter enhancer, globin, albumin, insulin and thelike.

Methods for producing and using genetic constructs and vectors are wellknown in the art and are described generally in Sambrook et al.,(supra), and Ausubel et al., Current Protocols in Molecular Biology,Greene Publishing, 1987. Methods for transforming selected host cellswith the vectors are also known, for example, the calcium chloridetreatment described by Cohen, S N; PNAS 69, 2110, 1972.

For a general discussion of constructs, promoters, enhancers, and hostcells, see Principles of Gene Manipulation and Genomics; Primrose, S etal., Blackwell Publishing 2006, Ed. 7., and From Genes to Genomes:Concepts and Applications of DNA Technology, Dale, J et al.,Wiley-Interscience, 2007, Ed. 2.

Host cells comprising the genetic constructs and vectors described maybe derived from prokaryotic or eukaryotic sources, for example yeast,bacteria, fungi, insect (eg baculovirus), animal, mammalian or plantorganisms. In one embodiment the host cells are isolated host cells.Prokaryotes most commonly employed as host cells are strains of E. coli.Other prokaryotic hosts include Pseudomonas, Bacillus, Serratia,Klebsiella, Streptomyces, Listeria, Saccharomyces, Salmonella andMycobacteria but are not limited thereto.

Eukaryotic cells for expression of recombinant protein include but arenot limited to Vero cells, HeLa, CHO (Chinese Hamster ovary cells), 293,BHK cells, MDCK cells, and COS cells as well as prostate cancer celllines such as PrEC, LNCaP, Du 145 and RWPE-2. The cells are availablefrom ATCC, Virginia, USA.

Prokaryotic promoters compatible with expression of nucleic acidmolecules of the invention include known art constitutive promoters(such as the int promoter of bacteriophage lambda and the bla promoterof the beta-lactamase gene sequence of pBR322) and regulatable promoters(such as lacZ, recA and gal). A ribosome binding site upstream of thecoding sequence may also be required for expression.

Host cells comprising genetic constructs, such as expression constructs,are useful in methods for recombinant production of polypeptides. Suchmethods are well known in the art (see for example Sambrook et al.supra). The methods commonly involve the culture of host cells in anappropriate medium in conditions suitable for or conducive to,expression and selection of a polypeptide of the invention. Cells with aselectable marker may additionally be grown on medium appropriate forselection of host cells expressing a polypeptide of the invention.Transformed host cells expressing a polypeptide of the invention areselected and cultured under conditions suitable for expression of thepolypeptide. The expressed recombinant polypeptide, may be separated andpurified from the culture medium using methods well known in the artincluding ammonium sulfate precipitation, ion exchange chromatography,gel filtration, affinity chromatography, electrophoresis and the like(e.g. Deutscher, Ed, 1990, Methods in Enzymology, Vol 182, Guide toProtein Purification). Host cells may also be useful in methods forproduction of a product generated by an expressed polypeptide of theinvention.

In another aspect, the present invention provides a method forpredicting, diagnosing or monitoring an acute cardiac disorder (ACD) ina subject, the method comprising:

measuring the level of an INS-SP biomarker in a biological sample takenor derived from the subject and comparing the level of said INS-SP withthe INS-SP biomarker level from a control or reference or referencerange wherein a measured level of INS-SP biomarker higher than thecontrol or reference level is indicative of ACD.

In another aspect the invention provides a method for monitoring aresponse to treatment of a an acute cardiac disorder (ACD) in a subject,the method comprising measuring the level of INS-SP biomarker in abiological sample from the subject and comparing the level of saidINS-SP biomarker with the INS-SP level from a control, reference, orreference range, wherein a change in the measured level of INS-SPbiomarker from the control or reference level is indicative of aresponse to the treatment.

It is known in the art that BNP precursors such as proBNP27-102proBNP27-47, can be used in predicting or diagnosing a cardiactransplant rejection episode and to distinguish between pulmonary andcardiovascular causes of dyspnea (shortness of breath). See US2005/0244902. It is contemplated that INS-SP can be used as an earlymarker of cardiac transplant rejection based on cardiac tissue analysis,and to distinguish pulmonary from acute cardiac disorders.

Accordingly, the invention also provides a method for predicting,diagnosing or monitoring a cardiac transplant rejection episode in asubject, the method comprising measuring the level of an INS-SPbiomarker in a biological sample from a subject after heart transplantand comparing the level of said INS-SP biomarker with the INS-SP levelfrom a control, reference or reference range, wherein a measured levelof an INS-SP biomarker higher than a control or reference level isindicative of transplant rejection.

In one embodiment, the invention provides a method for predicting,diagnosing or monitoring an acute cardiac disorder (ACD), cardiactransplant rejection, or ACD/pulmonary disorder in a subject, the methodcomprising measuring the level of an INS-SP biomarker in a biologicalsample from the subject within the first about two hours of onset of, orclinical presentation with ACD, cardiac transplant rejection orACD/pulmonary disorder. A measured level of an INS-SP biomarker iscompared with the INS-SP biomarker level from a control, reference orreference range wherein a measured level of the INS-SP biomarker higherthan the control or reference level is indicative of ACD or transplantrejection.

The skilled reader will appreciate that for evaluation purposes, theINS-SP biomarker level will generally be correlated with a referencevalue or range or a control value.

As used herein a control can be an individual or group from which INS-SPbiomarker samples are taken and a mean INS-SP biomarker leveldetermined. Usually, the individual or group will comprise normalhealthy individuals or a group of individuals not known to be sufferingfrom a biological event to be monitored, such as glucose handlingdisorders, diabetes, ACD (including cardiac transplant rejection), orACD/pulmonary disorder. INS-SP biomarker levels in most individuals arebetween 0.5-40 pmol/L, and the mean control level is about 9 pmol/L.Alternatively, the control level may be assessed based on a plurality ofreadings from previously tested individuals or groups.

Another example of a control level is a ratiometric measure between anINS-SP biomarker and insulin levels in cardiac tissue or tissue from adiabetic or individual with a glucose handling disorder. The subject'sINS-SP biomarker level(s) can be compared to the mean INS-SP biomakerlevel for that control population. The INS-SP level in the cardiactissue control population may be in the order of about 1.5 to 3,commonly about 2 to 3 or about 2.5 to 3 times (or more) higher thanINS-SP levels in the normal control population. The INS-SP level in thediabetic, or glucose handling disorder control population may be in theorder of about two to three times lower or higher (depending on thenature of the diabetes) than the INS-SP levels in the normal controlpopulation.³¹ Alternatively, the control may be one or more readings orthe mean of such readings taken from the same subject at an earliertime. Ascertaining appropriate controls and control levels forparticular methods is well known in the art.

It will be appreciated that the step of measuring INS-SP biomarkerlevels in a sample may be a single measurement on a single sample, orrepeated measurements on a number of samples depending on the biologicalevent being studied. In the case of ACD, measurement may comprise, forexample, 1 to 20 measurements of an INS-SP biomarker, 1 to 10, 1 to 5, 1to 3, 1 or 2, or 2 or 3 measurements, in samples taken or derived from asubject at different times. In one embodiment measurements are takenwithin about the first six, five, four, three, two hours, or within onehour of, onset of or clinical presentation with a disorder. Single, orrepeated measurements outside the sample period above may also be takento establish whether the INS-SP level biomarker has risen or fallencompared to the normal control level, or cardiac tissue control level,or related reference levels or ranges.

In one embodiment, the method comprises measuring INS-SP biomarkerlevels in 1 or 2 samples taken within about the first hour of onset orpresentation, followed by measuring INS-SP biomarker levels in 1 or 2samples taken within about two to about four hours, or about two toabout three hours of onset or presentation, or initial measurement ofthe INS-SP level.

As noted above, INS-SP levels measured within the first six, four, ortwo hours of onset or presentation, and can be five to fifteen timeshigher than INS-SP biomarker levels measured in a normal control.

In another embodiment, a level of an INS-SP biomarker in the sample inthe range about 40 to about 350 pmol/L, or about 45 to about 300 pmol/L,about 50 to about 250 pmol/L, or about 55 to about 200 pmol/L isindicative of ACD, cardiac transplant rejection, or distinguishes ACDfrom a pulmonary disorder.

In the case of a biological event such as diabetes, for example, orglucose handling disorders, measurement may comprise multiplecalculations in conjunction with established clinical assessment, suchas regularly used for insulin.

The biological sample as defined above can be any biological material inwhich an INS-SP biomarker can be located or secreted. In one embodimenta biological sample is a circulatory biological sample, for exampleblood, serum or plasma. In one embodiment, the biological sample iscardiac tissue.

Nucleic Acid Assays

The presence of INS-SP and its level of expression in the sample may bedetermined according to methods known in the art such as SouthernBlotting, Northern Blotting, FISH or quantitative PCR to quantitate thetranscription of mRNA [(Thomas, Proc. Nat, Acad. Sci. USA 77: 5201-52051980), (Jain K K, Med Device Technol. 2004 May; 15(4):14-7)], dotblotting, (DNA analysis) or in situ hybridization using an appropriatelylabelled probe, based on the sequences provided herein.

Accordingly, the invention also provides an assay for detecting thepresence of a nucleic acid molecule of the invention, in a sample, themethod comprising:

-   -   (a) contacting the sample with a polynucleotide probe which        hybridises to the nucleic acid sequence under stringent        hybridisation conditions; and    -   (b) detecting the presence of a hybridisation complex in the        sample.

In one embodiment the nucleic acid molecule is SEQ ID NO:17 or SEQ IDNO:19 or a variant or fragment thereof.

In one embodiment, the hybridisation probe is a labelled probe. Examplesof labels include fluorescent, chemiluminescent, radioenzyme andbiotin-avidin labels. Labelling and visualisation of labelled probes iscarried out according to known art methods such as those above.

For convenience the nucleic acid probe may be immobilized on a solidsupport including resins (such as polyacrylamides), carbohydrates (suchas sepharose), plastics (such as polycarbonate), and latex beads but notlimited thereto.

As discussed above the nucleic acid molecule probe may preferably be anRNA, cDNA or DNA molecule. In one embodiment the probe is, or includesSEQ ID NOs: 17 and 19.

Stringent hybridisation conditions are as discussed above.

The expression level of the nucleic acid marker may be determined usingknown art techniques such as RT-PCR and electrophoresis techniquesincluding SDS-PAGE. Using these techniques the DNA or cDNA sequence of anucleic acid molecule of the invention, in a subject sample isamplified, and the level of DNA or cDNA or RNA measured.

In an alternate method the DNA, cDNA or RNA level may be measureddirectly in the sample without amplification.

In one embodiment the method is Northern blot hybridization analysis.Probes for use in Northern blot hybridization analysis may be preparedbased on the INS-SP biomarker sequences identified herein. In oneembodiment, a probe includes at least 10, 12, 15, 18, 21, 24, 27, 30,36, 42, 51, 60, 63, 66, 69, 70 or 72 or more contiguous nucleotides of areference sequence.

Alternatively, the expression level may be measured using reversetranscription based PCR (RT-PCR) assays using primers specific for thenucleic acid sequences. If desired, comparison of the level of theINS-SP biomarker polynucleotide in the sample can be made with referenceto a control nucleic acid molecule the expression of which isindependent of the parameter or condition being measured. A controlnucleic acid molecule refers to a molecule in which the level does notdiffer between the disorder or transplant rejection state and thehealthy state. Levels of the control molecule can be used to normaliselevels in the compared populations. An example of such a controlmolecule is GAP-DH. The INS-SP biomarker polynucleotide of theinvention, will change levels with the biological event or disorder.

Peptide Assays

In one embodiment the measuring step comprises detecting binding betweenan INS-SP biomarker and a binding agent that binds, (includingselectively or specifically binds) INS-SP or a fragment or variantthereof. As a pre-step in the measurement an INS-SP biomarkerpolypeptide may be bound with a binding agent that binds INS-SP or afragment or variant thereof.

Accordingly, in one embodiment the invention provides an assay for anINS-SP biomarker in a biological sample, the assay comprising detectingand measuring the level of an INS-SP biomarker in the sample using anyknown methods.

In one embodiment, the biological sample is obtained from a subjectwithin six or four hours from onset of ACD, cardiac transplantrejection, or ACD/pulmonary disorder or within four hours of clinicalpresentation with ACD, cardiac transplant rejection, or ACD/pulmonarydisorder.

In one embodiment, the invention provides an assay for an INS-SPbiomarker comprising:

-   -   (a) binding one or more INS-SP biomarker polypeptides from a        biological sample; and    -   (b) measuring the level of bound INS-SP biomarker polypeptide.

In one embodiment, the INS-SP biomarker polypeptide is selected from thegroup INS-SP 1-9, and INS-SP 15-24, or a variant or fragment thereof. Itwill be appreciated that more than one type of INS-SP polypeptide may bebound in the assay, for example INS-SP 1-9 and INS-SP 15-24.

In one embodiment, the INS-SP biomarker polypeptide is bound using abinding agent. The binding agent is a selective (specific) bindingagent. That is, it has low cross-reactivity with other markers ofbiological events, and more particularly insulin. The binding agent inone embodiment is an antibody or antigen-binding fragment thereof. Wherean antibody is used in the assay, the antibody may be raised against anyantigenic part of the INS-SP biomarker, including at the N-terminal(1-9) or C-terminal (15-24) or INS-SP. In one embodiment the antibody israised against INS-SP 1-24, 1-9 or 15-24 (SEQ ID NOs: 14, 16 and 18) oran amino acid sequence encoded by a nucleotide sequence of theinvention; or a variant or fragment thereof.

The present invention also relates to such binding agents, antibodies,and antigen-binding fragments of the antibodies and their uses. Usesinclude in an assay, or in the manufacture of an assay, prognostic,diagnostic or monitoring tool for INS-SP biomarker. The assay or toolmay be used to monitor a biological event or disorder in a subjectincluding a glucose handling disorder, diabetes and ACD.

The antibodies may be in isolated or purified form. An antibody thatbinds to INS-SP or a fragment or variant thereof may be in any form,including all classes of polyclonal, monoclonal, bispecific, singlechain, human, humanized antibodies and chimeric antibodies produced bygenetic recombination. Also included is antiserum obtained by immunizingan animal such as a mouse, rat or rabbit with INS-SP or a fragment orvariant thereof. The antibodies may bind to a common INS-SP sequence ina group of INS-SP fragments, or to a specific INS-SP fragment, or evento sets of INS-SP fragments.

A fragment of an antibody or a modified antibody may also be used hereinso long as it binds BNP-SP or a fragment or variant thereof. Theantigen-binding fragment may be Fab, F(ab′), F(ab′), an Fc or Fvfragment or single chain Fv (scFv), in which Fv fragments from H and Lchains are ligated by an appropriate linker (Huston et al. Proc. Natl.Acad. Sci. USA. 85:5879-83 (1988)). The “Fc” portion of an antibodyrefers to that portion of an immunoglobulin heavy chain that comprisesone or more heavy chain constant region domains; CH1, CH2 and CH3, butdoes not include the heavy chain variable region.

The “Fv” portion of an antibody is the minimum antibody fragment thatcontains a complete antigen-recognition and antigen binding site. Theregion consists of a dimer of one heavy chain and one light chainvariable domain in tight, non-covalent association.

Fab fragments contain the constant domain of the light chain and thefirst constant domain (CHI) of the heavy chains. Fab′ fragments have afew residues added to the Fab carboxy terminus of the CHI domainincluding one or more cysteines from the antibody hinge region. F(ab′)₂fragments represent pairs of Fab′ fragments with cysteine hinges betweenthem, that have been separated. The F(ab′)₂ fragment has two-antigenbinding sites. Fab fragments may be produced by papain digestion ofantibodies.

For a discussion of antibodies and fragments see for example PNAS USA81: 6851-6855 (1984), Protein Eng 8(10) 1057-1062 (1995); ThePharmacology of Monoclonal Antibodies, vol. 113, Springer-verlag 1994,Rosenburg and Moore Eds; PNAS USA 90: 6444-6448 (1993); Nature 321:522-525 (1986); Nature 332: 323-329 (1988), and WO 2005/003154.

Methods for preparing antibodies, and detecting, modifying and isolatingsame are well known in the art (see for example Maintaining and usingAntibodies: A Practical Handbook, Howard, G et al., CRC Press 2006;Protein-protein Interactions: A Molecular Cloning Manual, Golemis E(Ed), CSHL Press, 2002; Harlow and Lane (1998,¹¹ Milstein¹⁸, Suresh¹⁹,and Brennan²⁰). In one embodiment antibodies used are produced byimmunizing a suitable host mammal. Fusion proteins comprising INS-SPbiomarkers may also be used as immunogens.

An antibody may be modified by conjugation with a variety of molecules,such as polyethylene glycol (PEG), biotin, streptavidin, andchemiluminescent, fluorescent, calorimetric; and radioimmunometriclabels as discussed herein. The modified antibody can be obtained bychemically modifying an antibody. These modification methods areconventional in the field.

Alternatively, an antibody may be obtained as a chimeric antibody,between a variable region derived from nonhuman antibody and theconstant region derived from human antibody, or as a humanized antibody,comprising the complementarity determining region (CDR) derived fromnonhuman antibody, the frame work region (FR) derived from humanantibody, and the constant region. Such antibodies can be prepared usingknown art methods.^(16,17,22)

In brief, methods of preparing polyclonal antibodies are known to theskilled artisan. Polyclonal antibodies can be raised in a mammal, forexample, by one or more injections of an immunizing agent and, ifdesired, ari adjuvant. Typically, the immunizing agent and/or adjuvantwill be injected in the mammal by multiple subcutaneous orintraperitoneal injections. The immunizing agent may include INS-SP or afragment or variant thereof or a fusion protein thereof. It may beuseful to conjugate the immunizing agent to a protein known to beimmunogenic in the mammal being immunized. Examples of such immunogenicproteins include but are not limited to keyhole limpet hemocyanin,bovine serum albumin, bovine thyroglobulin, and soybean trypsininhibitor. Examples of adjuvants which may be employed include Freund'scomplete adjuvant and MPL TDM adjuvant (monophosphoryl Lipid A,synthetic trehalose dicorynomycolate). The immunization protocol may beselected by one skilled in the art without undue experimentation.

Monoclonal antibodies may be prepared using hybridoma methods well knownin the art. See for example Kohler and Milstein, 1975¹¹, U.S. Pat. No.4,196,265, U.S. Pat. No. 4,816,567 and Golemis (surpra). The hybridomacells may be cultured in a suitable culture medium, alternatively, thehybridoma cells may be grown in vivo as ascites in a mammal. Preferredimmortalized cell lines are murine myeloma lines, which can be obtained,for example, from the American Type Culture Collection, Virginia, USA.Immunoassays may be used to screen for immortalized cell lines whichsecrete the antibody of interest. Sequences of INS-SP or fragments orvariants thereof may be used in screening.

Accordingly, also contemplated herein are hybridomas which areimmortalized cell lines capable of secreting a INS-SP specificmonoclonal antibody.

Well known means for establishing binding specificity of monoclonalantibodies produced by the hybridoma cells include immunoprecipitation,radiolinked immunoassay (RIA), enzyme-linked immunoabsorbent assay(ELISA) and Western blot. (Lutz et al., Exp. Cell. Res. 175:109-124(1988), Golemis (supra), and Howard (supra)). For example, the bindingaffinity of the monoclonal antibody can, for example, be determined bythe Scatchard analysis described in Munson et al., Anal Biochem 107: 220(1980). Samples from immunised animals may similarly be screened for thepresence of polyclonal antibodies.

Monoclonal antibodies can also be obtained from recombinant host cells.DNA encoding the antibody can be obtained from a hybridoma cell line.The DNA is then placed into an expression vector, transfected into hostcells (eg, COS cells, CHO cells, E. coli cells) and the antibodyproduced in the host cells. The antibody may then be isolated and/orpurified using standard techniques.

Other known art techniques for monoclonal antibody production such asfrom phage libraries, may also be used. See for example, Nature 352:624-628 (1991).

To facilitate detection, antibodies and fragments herein may be labelledwith detectable markers such as fluorescent, bioluminescent, andchemiluminescent compounds, as well as radioisotopes, magnetic beads andaffinity labels (e.g biotin and avidin). Examples of labels which permitindirect measurement of binding include enzymes where the substrate mayprovide for a coloured fluorescent product, suitable enzymes includehorseradish peroxidase, alkaline phosphatase, malate dehydrogenase andthe like. Fluorochromes (e.g Texas Red, fluorescein, phycobiliproteins,and phycoerythrin) can be used with a fluorescence activated cellsorter. Labelling techniques are well known in the art.

The monoclonal antibodies secreted by the cells may be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, reversephase HPLC, protein A-Sepharose, hydroxyapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography. See for example,Scopes, Protein Purification: Principles and Practice, Springer-Verlag,NY (1982).

The monoclonal antibodies or fragments may also be produced byrecombinant DNA means (see for example U.S. Pat. No. 4,816,567). DNAmodifications such as substituting the coding sequence for human heavyand light chain constant domains in place of the homologous murinesequences (U.S. Pat. No. 4,816,567 above) are also possible. Theantibodies may be monovalent antibodies. Methods for preparingmonovalent antibodies are well known in the art (U.S. Pat. Nos.5,334,708, 5,821,047, and 7,476,724). Production of chimeric (U.S. Pat.No. 4,816,567), bivalent antibodies (U.S. Pat. No. 5,843,708) andmultivalent antibodies are also contemplated herein (U.S. Pat. No.6,020,153).

Chimeric monoclonal antibodies are antibodies in which a portion of theheavy and/or light chain is identical with or homologous tocorresponding sequences in antibodies derived from a particular speciesor belonging to a particular antibody (sub)class. The remainder of thechain is identical, or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibody(sub)class, and fragments thereof, so long as they exhibit the requisitebiological activity. (See U.S. Pat. No. 4,816,567 supra).

The antibodies of the invention may further comprise humanizedantibodies or human antibodies. Humanized antibodies include humanimmunoglobulins in which residues from a complementary determiningregion (CDR) of the recipient are replaced by residues from a CDR of anon-human species. The production of humanized antibodies from non-humansources such as rabbit, rat and mouse are well known.^(13,14,15)

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries¹⁶; and transgenic methods,see, for example Neuberger 1996¹⁷; and Vaughan et al, 1998¹⁸.

Bispecific antibodies may also be useful. These antibodies aremonoclonal, preferably human or humanized, antibodies that have bindingspecificities for at least two different antigens. For example INS-SP ora variant or fragment thereof, and an antigen selected from the groupincluding preproinsulin, ANP, ANP-SP, BNP, CK-MB, TnT, TnI, BNP, BNP-SP,NT-BNP, myoglobin, LDH, aspartate aminotransferase, H-FABP, endothelin,adrenomedullin, renin, ischemia modified albumin and angiotensin II.Antibodies with greater than two specificities for example trispecificantibodies are also contemplated herein.

Methods for making bispecific antibodies are known in the art. See forexample Milstein and Cuello 1983¹⁹, Suresh et al., 1986²⁰ and Brennan etal., 1985²¹.

The INS-SP biomarker which is bound or selectively bound by the antibodyis INS-SP or a variant or fragment thereof as discussed above.

In one embodiment, the antibody binds the N-terminus (1-9) or C-terminus(15-24) of INS-SP. Examples of specific antigenic peptides which thebinding agent selectively binds include INS-SP (1-9) and INS-SP (15-24)SEQ ID NOs:16 and 18.

Binding of an INS-SP biomarker can be detected by any means known in theart including specific (antibody based) and non specific (such as HPLCsolid phase). Most commonly, antibodies herein are detected using anassay such as ELISA or RIA as noted above. Competitive binding assays,sandwich assays, non-competitive assays, fluoroimmunoassay,immunofluorometric assay, or immunoradiometric assays, luminescenceassays, chemiluniescence assays and mass spectrometry analysis such asurface-enhanced laser desorption and ionization (SELDI) electrosprayionization (ESI), matrix assisted laser-desorption ionization (MALDI),fourier transform Ion cyclotron resonance mass spectroscopy (FTICR)alone or in combination with non-specific binding agents such aschromatography formats are also feasible. See for example, Golemis, Eand Howard G. (supra).

Conveniently, an antibody can be fixed to a solid substrate tofacilitate washing and isolation of the INS-SP/antibody complex. Bindingof antibodies to a solid support can be achieved using known arttechniques. See for example Handbook of Experimental Immunology, 4thedition, Blackwell Scientific Publications, Oxford (1986). Useful solidsubstrates for antibodies include glass, nylon, paper and plastics.Similarly, INS-SP can be adsorbed onto a solid substrate such asadsorbent silica, or resin particles, or silicon chips optionally coatedor derivatised with ion exchange, reverse phase (eg C18 coating) orother materials. The substrate may be in the form of beads, plates,tubes, sticks or biochips. Examples of biochips include Ciphergen,ProteinChip arrays (Ciphergen Biosystems (CA, USA)), and PackardBioChips available from Perkin Elmer, USA. See also U.S. Pat. No.6,225,047, U.S. Pat. No. 6,329,209. The biochips may include achromatographic surface. Biochips or plates with addressable locationsand discreet microtitre plates are particularly useful. Also preferredfor use are multiplex systems where beads containing antibodies directedto multiple analytes are used to measure levels of the analytes in asingle sample. Analytes to be measured may include other cardiac markersas well as INS-SP or variants or fragments thereof. One example of asuitable multiplex bead system for use herein is the Luminex FlurokineMultianalyte Profiling system.

Antibody assay methods are well known in the art see for example U.S.Pat. No. 5,221,685, U.S. Pat. No. 5,310,687, U.S. Pat. No. 5,480,792,U.S. Pat. No. 5,525,524, U.S. Pat. No. 5,679,526, U.S. Pat. No.5,824,799, U.S. Pat. No. 5,851,776, U.S. Pat. No. 5,885,527, U.S. Pat.No. 5,922,615, U.S. Pat. No. 5,939,272, U.S. Pat. No. 5,647,124, U.S.Pat. No. 5,985,579, U.S. Pat. No. 6,019,944, U.S. Pat. No. 6,113,855,U.S. Pat. No. 6,143,576 and for unlabelled assays U.S. Pat. No.5,955,377, and U.S. Pat. No. 5,631,171 see also Zola, MonoclonalAntibodies: A Manual of Techniques pp 147-158 (CRC Press, Inc 1987),Harlow and Lane (1998) Antibodies, A Laboratory Manual, Cold SpringHarbour Publications, New York, and US 2005/0064511 for a description ofassay formats and conditions. All of the above references areincorporated herein by reference in their entirety.

Immunoassay analysers are also well known and include Beckman Access,Abbott AxSym, Roche ElecSys and Dade Behring Status systems amongstothers which are well described.²²

Binding of an INS-SP biomarker and an antibody to form a complex can bedetected directly or indirectly. Direct detection is carried out usinglabels such as fluorescence, luminescence, radionuclides, metals, dyesand the like. Indirect detection includes binding detectable labels suchas digoxin or enzymes such as horseradish peroxidase and alkalinephosphatase to form a labelled antibody followed by a step of detectingthe label by addition of detection reagents.

Horseradish peroxidase for example can be incubated with substrates suchas o-Phenylenediamine Dihyhydrochloride (OPD) and peroxide to generate acoloured product whose absorbance can be measured, or with luminol andperoxide to give chemiluminescent light which can be measured in aluminometer as is known in the art. Biotin or digoxin can be reactedwith binding agents that bind strongly to them. For example, theproteins avidin and streptavidin will bind strongly to biotin. A furthermeasurable label is then covalently bound or linked thereto either bydirect reaction with the protein, or through the use of commonlyavailable crosslinking agents such as MCS and carbodiimide, or byaddition of chelating agents.

Generally, the complex is separated from the uncomplexed reagents forexample by centrifugation. If the antibody is labelled, the amount ofcomplex will be reflected by the amount of label detected.Alternatively, an INS-SP biomarker may be labelled by binding to anantibody and detected in a competitive assay by measuring a reduction inbound labelled INS-SP biomarker when the antibody-labelled-INS-SPbiomarker is incubated with a biological sample containing unlabelledINS-SP biomarker. Other immunoassays may be used for example a sandwichassay.

In one embodiment, following contact with the antibody, usuallyovernight for 18 to 25 hours at 4° C., or for 1 to 2 to 4 hours at 25°C. to 40° C., the labelled INS-SP biomarker bound to the binding agent(antibody) is separated from the unbound labelled INS-SP biomarker. Insolution phase assays, the separation may be accomplished by addition ofan anti gamma globulin antibody (second-antibody) coupled to solid phaseparticles such as cellulose, or magnetic material. The second-antibodyis raised in a different species to that used for the primary antibodyand binds the primary antibody. All primary antibodies are thereforebound to the solid phase via the second antibody. This complex isremoved from solution by centrifugation or magnetic attraction and thebound labelled peptide measured using the label bound to it. Otheroptions for separating bound from free label include formation of immunecomplexes, which precipitate from solution, precipitation of theantibodies by polyethyleneglycol or binding free labelled peptide tocharcoal and removal from solution by centrifugation of filtration. Thelabel in the separated bound or free phase is measured by an appropriatemethod such as those presented above.

Competitive binding assays can also be configured as solid phase assaysthat are easier to perform and are therefore preferable to those above.This type of assay uses plates with wells (commonly known as ELISA orimmunoassay plates), solid beads or the surfaces of tubes. The primaryantibody is either adsorbed or covalently bound to the surface of theplate, bead or tube, or is bound indirectly through a second anti gammaglobulin or anti Fc region antibody adsorbed or covalently bound to theplate. Sample and labelled peptide (as above) are added to the plateeither together or sequentially and incubated under conditions allowingcompetition for antibody binding between INS-SP in the sample and thelabelled peptide. Unbound labelled peptide can subsequently be aspiratedoff and the plate rinsed leaving the antibody bound labelled peptideattached to the plate. The labelled peptide can then be measured usingtechniques described above.

Sandwich type assays have greater specificity, speed and greatermeasuring range. In this type of assay an excess of the primary antibodyto an INS-SP biomarker is attached to the well of an ELISA plate, beador tube via adsorption, covalent coupling, or an anti Fc or gammaglobulin antibody, as described above for solid phase competitionbinding assays. Sample fluid or extract is contacted with the antibodyattached to the solid phase. Because the antibody is in excess thisbinding reaction is usually rapid. A second antibody to an INS-SPbiomarker is also incubated with the sample either simultaneously orsequentially with the primary antibody. This second antibody is chosento bind to a site on the INS-SP biomarker that is different from thebinding site of the primary antibody. These two antibody reactionsresult in a sandwich with the INS-SP biomarker from the samplesandwiched between the two antibodies. The second antibody is usuallylabelled with a readily measurable compound as detailed above forcompetitive binding assays. Alternatively a labelled third antibodywhich binds specifically to the second antibody may be contacted withthe sample. After washing away the unbound material the bound labelledantibody can be measured and quantified by methods outlined forcompetitive binding assays.

A dipstick type assay may also be used. These assays are well known inthe art. They may for example, employ small particles such as gold orcoloured latex particles with specific antibodies attached. The liquidsample to be measured may be added to one end of a membrane or paperstrip preloaded with the particles and allowed to migrate along thestrip. Binding of the antigen in the sample to the particles modifiesthe ability of the particles to bind to trapping sites, which containbinding agents for the particles such as antigens or antibodies, furtheralong the strip. Accumulation of the coloured particles at these sitesresults in colour development are dependent on the concentration ofcompeting antigen in the sample. Other dipstick methods may employantibodies covalently bound to paper or membrane strips to trap antigenin the sample. Subsequent reactions employing second antibodies coupledto enzymes such as horse radish peroxidase and incubation withsubstrates to produce colour, fluorescent or chemiluminescent lightoutput will enable quantitation of antigen in the sample.

As discussed in the following examples, in one embodimentradioimmunoassay (RIA) is the laboratory technique used. In one RIA aradiolabelled antigen and unlabelled antigen are employed in competitivebinding with an antibody. Common radiolabels include ¹²⁵I, ¹³¹I, ³H and¹⁴C.

Radioimmunoassays involving precipitation of an INS-SP biomarker with aspecific antibody and radiolabelled antibody binding protein can measurethe amount of labelled antibody in the precipitate as proportional tothe amount of the INS-SP biomarker in the sample. Alternatively, alabelled INS-SP biomarker is produced and an unlabelled antibody bindingprotein is used. A biological sample to be tested is then added. Thedecrease in counts from the labelled INS-SP biomarker is proportional tothe amount of INS-SP biomarker in the sample.

In RIA it is also feasible to separate bound INS-SP biomarkers from freeINS-SP biomarkers. This may involve precipitating the INS-SPbiomarker/antibody complex with a second antibody. For example, if theINS-SP biomarker/antibody complex contains rabbit antibody then donkeyanti-rabbit antibody can be used to precipitate the complex and theamount of label counted. For example in an LKB, Gammamaster counter. SeeHunt et al.²²

The methods of the invention further comprise measuring the levels ofone or more other markers of glucose handling disorders, diabetes, ACD,cardiac transplant rejection, or ACD/pulmonary disorder, that are not anINS-SP biomarker. The level of the other marker or, markers can becompared to mean control levels from a control population. A deviationin the measured level from the mean control level is predictive ordiagnostic of a glucose handling disorder, diabetes or a predispositionthereto, ACD or cardiac transplant rejection.

While the methods of the invention have been described with respect to ahigher level or increase in INS-SP biomarker levels being indicative ofACD, or cardiac transplant rejection and a lower, different or deviatedlevel of INS-SP biomarker being indicative of diabetes or glucosehandling disorders, it is also possible that in some events or disordersthe levels of INS-SP biomarker(s) will fall or be lower or will rise orbe higher depending on the metabolic effect of the event or disorder.Measuring deviations above or below a control level are alsocontemplated.

Other markers which are particularly useful herein for ACD and cardiactransplant rejection include troponin, troponin T, troponin I, creatinkinase MB, myoglobin, BNP, NT-BNP, BNP-SP, BNP-SP fragments, ANP,ANP-SP, ANP-SP fragments, LDH, aspartate aminotransferase, H-FABP,endothelin, adrenomedullin, renin, ischemia modified albumin, andangiotensin II¹. These markers are all implicated in cardiac dysfunctionor disease. For diabetes, and glucose handling disorders, other markersinclude insulin, lactate, glucose, fatty acids and triglycerides ormarkers therefor. Assays for such markers are well known and used in theart. For example, various such assays are used routinely in clinicalsettings as described by Vogel, H, (2007) Drug Discovery and Evaluation:Pharmacological Assays Ed 3. Springer pp. Ed.: 3, pp 2071 and by Rungeet al. (2006) Principles of Molecular medicine Ed. 2 Springer, pp 1268.Kits and reagents for performing such assays are commercially availablefrom a number of suppliers including QuantiChrom™ and EnzyChrome™glucose, fatty acid and triglyceride assays (BioAssay Systems,California, USA) and Glucose, Triglyceride and Free Fatty Acid AssayKits (BioVision, California, USA). Correlating the level of INS-SP withother markers can increase the predictive, diagnostic or monitoringvalue of INS-SP. In the case of ACD, cardiac transplant rejection orACD/pulmonary disorder combining INS-SP marker levels with known cardiacmarkers can increase the predictive or diagnostic value of a patientoutcome.

Analysis of a number of peptide markers can be carried outsimultaneously or separately using a single test sample. Simultaneous,two or multi-site format assays are preferred. Multiplex bead,microassay or biochip systems are particularly useful. The beads, assaysor chips can have a number of discreet, often addressable locations,comprising an antibody to one or more markers including INS-SP andINS-SP fragments. The one or more markers include more than one INS-SPmarker. For example, it may be useful to assay for N-terminal andC-terminal INS-SP fragments and combine the assay results. Many othersuch marker combinations are feasible. US2005/0064511, U.S. Pat. No.6,019,944, and Ng and Ilang, J. Cell Mol. Med., 6:329-340 (2002) providea description of microarray, chips, capillary devices and techniquesuseful in the present invention. Luminex provides a multiplex beadsystem useful in the present invention. See also The Protein ProtocolsHandbook, supra. Laboratory analysers suitable for use with separate orsequential assays include AxSym (Abbott, USA), ElecSys (Roche), Access(Beckman), ADVIA CENTAUR® (Bayer) and Nichols Advantage® (NicholsInstitute) immunoassay system.

In one embodiment simultaneous assays of a plurality of polypeptides areperformed on a single surface such as a chip or array.

In another embodiment separate assays of one or more non-INS-SP markersare performed and the results collated or combined with INS-SP biomarkerresults.

Where a subject is to be monitored, a number of biological samples maybe taken over time. Serial sampling allows changes in marker levels,particularly INS-SP biomarkers to be measured over time. Sampling canprovide information on the approximate onset time of an event, theseverity of the event, indicate which therapeutic regimes may beappropriate, response to therapeutic regimes employed, or long termprognosis. Analysis may be carried out at points of care such as inambulances, doctors offices, on clinical presentation, during hospitalstays, in outpatients, or during routine health screening.

The methods of the invention may also be performed in conjunction withan analysis of one or more risk factors such as but not limited to age,weight, level of physical activity, sex and family history of eventssuch as diabetes, glucose handling disorders, and cardiac events. Testresults can also be used in conjunction with the methods of theinvention. For example, glucose tolerance tests, ECG results andclinical examination. A statistically significant change in circulatinglevel of INS-SP, together with one or more additional risk factors ortest results may be used to more accurately diagnose or prognose thesubject's condition.

The methods herein can also be used as a guide to therapy. For examplewhat therapies to initiate and when, therapy monitoring, detection ofpositive or adverse effects of therapy, for example heart toxicity ofantimitotic drugs, insulin, glucose handling, triglyceride and fattyacid concentrations, metformin and/or statin therapy, and adjustment oftherapeutic regimes if and when required dependent on results. This canimprove short, medium and long term outcomes for patients. For a guideto treatments see Troughton et al.⁸

Acute Cardiac Disorders

The applicants have shown that concentrations of INS-SP biomarker suchas INS-SP (1-9) are correlated with acute cardiac disorders. Moreover,INS-SP biomarker levels are at their highest upon clinical presentationin the case of patients presenting with suspected acute myocardialinfarction (AMI) or heart attack. Patients presenting with acute cardiacdisorders, and in particular acute cardiac ischemia coronary arterydisease caused by (heart attack leaving scarring in the heart muscle ormyocardium) may or may not experience subsequent myocardial infarction(MI). The group which does not experience MI can not be readilydiagnosed using current clinical techniques and markers. For the firsttime, the applicants have therefore provided a useful early and specificmarker for myocardial damage associated with MI. This may allow theearly diagnosis of myocardial damage due to adverse events (AEs) andallow a physician to distinguish such cases from other acute coronarysyndromes as well as from other causes of a chest pain. For exampleangina, gastro-intestinal disease, lung/pleural disorders and the like.This significantly shortens the window of 6 hours to 12 hours currentlyexperienced waiting for elevation of levels of current cardiacbiomarkers such as myoglobin, CK-MB, TnT and TnI. A more precisediagnosis and treatment can therefore be effected earlier, reducingmorbidity and mortality and giving better prognostic outcomes.

In another embodiment, the invention has application in monitoringreperfusion treatment in cardiac patients. Reperfusion treatmentcommonly includes percutaneous coronary intervention (eg angioplasty)and/or pharmacological treatment. Thrombolytic drugs forrevascularisation are commonly employed in pharmacological treatment.Adjunctive therapies include anticoagulant and anti-platelet therapies.Reperfusion treatment is most effective when employed as soon aspossible after diagnosis. INS-SP testing to accelerate diagnosis allowsprompt introduction of reperfusion treatment. Effectiveness of treatmentcan also be monitored by repeat testing, and therapy adjusted asappropriate. For a comprehensive discussion of reperfusion treatment seeBraunwald et al herein.

Cardiac Disease

The methods of the invention may also be useful to diagnose or predictcardiac disease in a subject.

Cardiac Transplant Rejection

The invention also has applications in monitoring heart transplant,commonly a cardiac allograft transplant, rejection through regulartissue biopsy during and after transplant using INS-SP biomarkermeasurements. An increase in INS-SP biomarker levels measured withinsix, four or two hours, of heart transplant relative to a control levelmay be predictive or diagnostic of a rejection episode.

The present invention also provides an assay for INS-SP biomarkers in abiological sample. In one embodiment the sample is obtained from asubject within about six, four or two hours from onset of, or withinabout six, four or two hours of clinical presentation with ACD, cardiactransplant rejection or ACD/pulmonary disorder. The assay comprisesdetecting and measuring the level of INS-SP biomarker in the sampleusing any known methods. In one embodiment, the assay is an in vitroassay. Such methods include all of the known assay techniques discussedabove as well as gel electrophoresis techniques, Western blot, gas phasespectroscopy, atomic force microscopy, surface plasmon resonance, massspectroscopy but not limited thereto²³.

In one embodiment the assay comprises one or more nucleic acid sequenceswhich bind to one or more of the INS-SP biomarker nucleic acid sequencesof the invention. A large range of sense and antisense probes andprimers can be designed from the nucleic acid sequences herein. Theexpression level of the INS-SP biomarker sequence is identified usingknown art techniques discussed above. The array can be a solid substratee.g., a “chip” as described in U.S. Pat. No. 5,744,305 or anitrocellulose membrane. For a discussion of useful arrays see forexample Microarray Technology and its Application, Müller, U et al.,Springer 2005, and Gene Expression Profiling by Microarrays: ClinicalImplications, Hofmann, W-K; Cambridge University Press 2006.

Proteins expressed by the INS-SP biomarker herein may also be used inassays, and results compared to expression levels of the same proteinexpressed in a normal control sample. Protein presence and quantity maybe assessed using assay formats known in the art and discussed herein.

The presence of INS-SP biomarker is preferably detected in the sample bybinding INS-SP biomarker to a binding agent such as an antibody of theinvention and measuring the presence of the amount of bound INS-SPbiomarker.

As noted above, antibodies which bind or selectively bind INS-SPincluding variants and fragments thereof, form a further aspect of theinvention and the antibodies may be prepared by the techniques discussedabove. The antibodies are useful in the methods and assays of theinvention.

In a further aspect, the invention provides a kit for predicting,diagnosing, assessing or monitoring a biological event in a subjectincluding glucose handling disorders, diabetes, acute cardiac disorderACD, (including cardiac transplant rejection), or ACD/pulmonarydisorder, comprising an INS-SP biomarker binding agent (or bindingagents for multiple INS-SP biomarkers) including an antibody orantigen-binding fragment of the invention. When the kit is for use indiagnosing ACD, cardiac transplant rejection, or an ACD/pulmonarydisorder, the biological sample is in one embodiment, for example,obtained from a subject within six, four or two hours of onset of, orclinical presentation with ACD, cardiac transplant rejection, orACD/pulmonary disorder.

The invention also provides a kit for predicting, diagnosing, assessingor monitoring an acute cardiac disorder (ACD), cardiac transplantrejection, or an ACD/pulmonary disorder comprising a binding agent ofthe invention, wherein the kit is calibrated to measure INS-SP levels inthe range of about 0.1 to about 500 pmol/L, preferably about 1 to about300 pmol/L, preferably about 10 to about 250 pmol/L.

Calibration of assays can be effected according to known art techniques,for example using blood samples with known levels of INS-SP biomarker,or a set of calibrates with different known levels of INS-SP in each.Test strips for use in diagnostic kits are commonly calibrated duringmanufacture. See for example U.S. Pat. No. 6,780,645. The kit is usefulfor measuring the level of INS-SP biomarker in a biological sample. Thedetection reagents may be oligonucleotide sequences complementary toINS-SP or a fragment of the INS-SP marker, or antibodies which bind tothe polypeptides encoded by the marker. The reagents may be bound to asolid matrix as discussed above or packaged with reagents for bindingthem to the matrix. The solid matrix or substrate may be in the form ofbeads, plates, tubes, dip sticks, strips or biochips all as discussedabove.

Detection reagents include wash reagents and reagents capable ofdetecting bound antibodies (such as labelled secondary antibodies), orreagents capable of reacting with the labelled antibody.

The kit will also conveniently include a control reagent (positiveand/or negative) and/or a means for detecting the nucleic acid,polypeptide, or antibody. Instructions for use may also be included withthe kit, such as taking a biological sample from a subject within six,four or two hours of onset or presentation with ACD, cardiac transplantrejection or ACD/pulmonary disorder, measuring the level of INS-SP inthe sample, comparing same to a control level and associating the resultwith cardiac status. Generally an increase in the INS-SP marker levelfrom a control is indicative of ACD or cardiac transplant rejection, orACD as opposed to a pulmonary disorder.

In the case of diabetes a lower or higher INS-SP biomarker marker levelfrom a control is indicative of diabetes or a predisposition to same,whether it is higher or lower depending on the nature of the diabetesand the diabetic status of the subject.

Most usually, the kits will be formatted for assays known in the art,and in one embodiment for PCR, Northern hybridization or Southern ELISAassays, as are known in the art.

The kits may also include one or more additional assays for markers forACD, transplant rejection, or ACD/pulmonary disorders. In the case ofACS the additional marker assay may include an assay or assays for oneor more of troponin, troponin T, troponin I, creatin kinase MB,myoglobin, BNP, BNP-SP, BNP-SP fragments, ANP, ANP-SP, ANP-SP fragments,NT-BNP, LDH, aspartate aminotransferase, H-FABP, endothelin,adrenomedullin, ischemia modified albumin, renin and angiotensin II. Inone embodiment all of the markers are included in the kit.

In the case of diabetes the additional kit components may be measurementmeans for markers that may include insulin, glucose, lactate,triglycerides and fatty acids or markers therefore.

The kit will be comprised of one or more containers and may also includecollection equipment, for example, bottles, bags (such as intravenousfluids bags), vials, syringes, and test tubes. At least one containerholds a product which is effective for predicting, diagnosing, ormonitoring a biological event such as diabetes, ACD (particularly ACS),transplant rejection, or ACD/pulmonary disorder. The product is usuallya nucleic acid molecule, polypeptide or a binding agent, particularly anantibody or antigen-binding fragment of the invention, or a compositioncomprising any of these. In a preferred embodiment, an instruction orlabel on, or associated with, the container indicates that thecomposition is used for predicting, diagnosing, or monitoring thebiological event. Other components may include needles, diluents andbuffers. Usefully, the kit may include at least one container comprisinga buffer, such as phosphate-buffered saline, Ringer's solution anddextrose solution.

Binding agents that bind or selectively bind an INS-SP biomarker (and,optionally, a non-INS-SP biomarker) are desirably included in the kit.In one embodiment, the binding agent is an antibody, preferably anantibody or antigen-binding fragment of the invention. The antibody usedin the assays and kits may be in one embodiment a monoclonal orpolyclonal and may be prepared in any mammal as discussed above. Theantibodies may be prepared against a native peptide encoded or indicatedby a INS-SP biomarker nucleic acid sequence of the invention, INS-SP(1-24), INS-SP (1-9), INS-SP (15-24), or a synthetic peptide based on,or including same, or may be raised against an exogenous sequence fusedto a nucleic acid sequence encoding an INS-SP biomarker peptide of theinvention.

In one kit embodiment an INS-SP biomarker detection reagent isimmobilized on a solid matrix such as a porous strip or chip to form atleast one INS-SP biomarker detection site. The measurement or detectionregion of the porous strip may include a plurality of detection sites,such detection sites containing an INS-SP biomarker detection reagent.The sites may be arranged in a bar, cross or dot or other arrangement. Atest strip or chip may also contain sites for negative and/or positivecontrols. The control sites may alternatively be on a different strip orchip. The different detection sites may contain different amounts ofimmobilized nucleic acids or antibodies eg, a higher amount in the firstdetection site and lower amounts in subsequent sites. Upon the additionof a test biological sample the number of sites displaying a detectablesignal provides a quantitative or semi-quantitative indication of theamount of INS-SP biomarker present in the sample.

Also included in the kit may be a device for sample analysis comprisinga disposable testing cartridge with appropriate components (markers,antibodies and reagents) to carry out sample testing. The device willconveniently include a testing zone and test result window.Immunochromatographic cartridges are examples of such devices. See forexample U.S. Pat. No. 6,399,398; U.S. Pat. No. 6,235,241 and U.S. Pat.No. 5,504,013.

Alternatively, the device may be an electronic device which allowsinput, storage and evaluation of levels of the measured marker againstcontrol levels and other marker levels. US 2006/0234315 providesexamples of such devices. Also useful in the invention are Ciphergen'sProtein Chip® which can be used to process SEMI results usingCiphergen's Protein Chip® software package.

In this specification where reference has been made to patentspecifications, other external documents, or other sources ofinformation, this is generally for the purpose of providing a contextfor discussing the features of the invention. Unless specifically statedotherwise, reference to such external documents is not to be construedas an admission that such documents;

or such sources of information, in any jurisdiction, are prior art, orform part of the common general knowledge in the art.

The invention will now be illustrated in a non-limiting way by referenceto the following examples.

Example 1

Methods

All human protocols were approved by the Upper South Regional EthicsCommittee of the Ministry of Health, New Zealand and were performed inaccord with the Declaration of Helsinki.

Chemicals

Synthetic human INS signal peptides INS-SP (1-9) and INS-SP (15-24) (SEQID NOs:16 and 18) were synthesised by Mimotopes (Australia) using a mildFmoc Solid Phase Synthesis method³⁰.

All buffer reagents were purchased from BDH® (UK) and/or Sigma (Mo,USA). INS-SP (1-9) and INS-SP (15-24) were synthesised with cysteine fordirectional carrier coupling. Both peptides were also synthesised with atyrosyl residue for tracer preparation on the same peptide.

Human Studies

For the healthy volunteer reference range study, venous blood sampleswere obtained from 20 healthy volunteers (13 woman, average age 48.8±3.2years (range 21-72 years), BMI 25.9±1.0 kg/m²) after an overnight fast.Samples were taken into tubes on ice and centrifuged at +4.0° C. at 2700g for 5 minutes and the plasma stored at −80° C. until analysed.

For analysis of INS-SP biomarker concentrations in acute cardiac injury,we studied 9 consecutive patients (4 woman, average age 70±8 years(range 59-79 years)), presenting to the Coronary Care Unit atChristchurch Hospital within 6 hours of the onset of chest pain andclear evidence of ST-elevation acute MI, together with a rise then fallin plasma troponin T (TnT). Patients with cardiogenic shock wereexcluded. All nine patients had an ECG during the hospital stay. Thetime between the onset of chest pain and drawing of the baseline (time0) venous sample was 3.7±0.2 hours. An 18-gauge intravenous cannula wasinserted into a forearm vein for blood sampling. Venous samples (10 ml)were drawn on admission to the Coronary Care Unit (time 0) andthereafter at 0.5, 1, 2, 4, 8, 12, 24 and 72 hours as in-patients.Samples were taken into tubes on ice and centrifuged at +4° C. at 2700 gfor 5 min and the plasma stored at −80° C. until analysed.

Plasma Extraction

All plasma samples were extracted on SepPak Cartridges, (Waters, USA) aspreviously described²², dried and stored at −20° C. prior to RIA andHPLC.

INS-SP RIA

For the measurement of putative human INS-SP biomarker peptides, wegenerated novel IR RIA′s directed against amino acids INS-SP 1-9 (SEQ IDNO:16) and 15-24 (SEQ ID NO:18) of the human preproinsulin (1-24) signalsequence (SEQ ID NO:14).

Antibody Generation

preproINS(1-9)^(Cys10) and (15-24)^(Cys14) were coupled to malemidetreated N-e-maleimidocaproyloxy succinimide ester (EMCS) derivatised BSAin PBS (pH 7.0) by gentle mixing at room temperature. Coupled peptidewas emulsified with Freund's adjuvant (2 ml) and injected subcutaneously(2 ml total) in 2 New Zealand white rabbits over 4-5 sites at monthlyintervals. Rabbits were bled 12 days after injection to assess antibodytitres until adequate levels were achieved. For RIA, INS-SP IR wasdetermined using antiserum at a final dilution of 1:30,000.

Iodination and Assay Method

preproINS (1-9) and (15-24) with coupled tyrosyl residues were iodinatedvia the Chloramine T method and purified on reverse phase HPLC (RP-HPLC)as previously described²¹. From this preparation an iodinated tracerform after RP-HPLC were tested. All samples, standards, radioactivetraces and antiserum solutions were diluted in potassium based assaybuffer.²² The assay incubate consisted of 100 μL sample or standard(0-640 pmol human preproINS (1-10) or (15-24) combined with 100 μLantiserum which was vortexed and incubated at 4° C. for 24 hours. 100 μLof trace (4000-5000 cpm) was then added and further incubated for 24hours at 4° C. Free and bound immunoreactivities were finally separatedby solid phase second antibody method (donkey anti-sheep Sac-Cela, IDSLtd, England) and counted in a Gammamaster counter (LKB, Uppsala,Sweden).

Statistical Analysis

All results are presented as mean±SEM. Time-course data were analysedusing two-way ANOVA for repeated measurements followed by leastsignificant difference post-hoc testing. Correlation analysis of plasmahormone concentrations was carried out using a general linear regressionmodel. In all analyses, a P-value <0.05 was considered significant.

Results

To determine if the 24 amino acid signal peptide of insulin, orfragments derived from it, are present in circulation of humans, wedeveloped a specific radioimmunoassay (RIA) directed against residues1-9 and 15-24 of preproinsulin(1-24). Dilution of plasma extractsdemonstrate parallelism with the standard curve (not shown). Plasmaconcentrations of INS-SP biomarker in healthy humans were 8.8±2.6 pmol/L(n=20) (FIG. 1).

Having established that IR INS-SP (1-9) peptides are present in humanplasma we then measured serial concentrations of IR INS-SP in patientswith documented AMI (n=9, FIG. 2). Highest concentrations of IR INS-SPwere observed at hospital admission and slowly dropped to stable levelsover 12 to 72 hours. Importantly, average peak levels at admission werefive to fifteen-fold higher than levels in normal healthy volunteers.

Preferably, IR INS-SP fragments are detected.

Example 2

Six patients with clinically stable suspected ACS were catheterized andblood samples from multiple organ sites: these were the femoral arteryFA(1) and FA(2) femoral vein (FV), renal vein (RV), hepatic vein (HV),inferior vena cava (IVC), jugular (JUG), cardiac coronary sinus vein(CS) and pulmonary artery (PA). Blood was collected into chilled EDTAtubes, prepared from plasma by centrifugation and the plasma submittedto INS-SP RIA. FIG. 2 shows the highest sites of INS-SP biomarkerconcentration are the jugular, renal and femoral veins.

Example 3

To assess the role that INS-SP may have in the control of metabolismand/or energy balance, 7 normal healthy volunteers were given 75 g oralglucose. As can be seen in FIG. 3, plasma INS-SP biomarker levels weresignificantly decreased after ingestion of glucose, consistent with ithaving a role in energy balance. In contrast, plasma concentrations ofInsulin were significantly increased after glucose ingestion, stronglysuggesting points of contrast between the two peptides in control ofenergy balance.

Conclusion

Circulating INS-SP biomarker concentrations in clinically stablepatients are likely derived from jugular, renal or peripheral sources.The increase of INS-SP peptides and subpeptides in response todocumented AMI support the idea that they have a role as a biomarker ofmetabolic and cardiac disease. The response of INS-SP biomarker plasmalevels to increases in plasma glucose also suggests it may have a rolein energy balance.

Discussion

This evidence is the first to document the signal peptide ofprepro-insulin, and fragments thereof, as being present in thecirculation and extracellular space. We show in the first instance thatthe measurement of INS-SP IR in blood has potential as a rapid biomarkerof acute cardiac ischemia and/or subsequent injury and in the secondinstance, that measurement of INS-SP after the event has potential meritas a marker of long term prognosis and outcome.

We also show that measurement in plasma of an INS-SP biomarker haspotential use in the arena of metabolism and/or energy balance,especially in the assessment of glucose handling.

Those skilled in the art will of course appreciate that the abovedescription is provided by way of example and that the invention is notlimited thereto.

REFERENCES

-   1. Braunwald E, Zipes D P, Libby P. Acute myocardial infarction Chp.    35 Heart disease: a textbook of cardiovascular medicine, 6^(th)    ed. 2001. pgs. 1114-1231.-   2. Richards A M, Nicholls M G, Yandle T G, Frampton C, Espiner E A,    Turner J G, Buttimore R C, Lainchbury J G, Elliott J M, Ikram H,    Crozier I G, Smyth D W. Plasma N-terminal pro-brain natriuretic    peptide and adrenomedullin: new neurohormonal predictors of left    ventricular function and prognosis after myocardial infarction.    Circulation 1998 97:1921-1929.-   3. Jernberg T, Stridsberg M, Venge P, Lindahl B. N-terminal pro    Brain Natriuretic Peptide on admission for early risk stratification    of patients with chest pain and no ST-segment elevation. J. Am.    Coll. Cardiology 2002 40:437-445.-   4. Omland T, Persson A, Ng L, O'Brien R, Karlsson T, Herlitz J,    Hartford M, Caidahl K. N-terminal pro-B-type natriuretic peptide and    long-term mortality in acute coronary syndromes. Circulation. 2002    106:2913-2918.-   5. Pemberton C J, Johnson M L, Yandle T G, Espiner E A.    Deconvolution Analysis of the Secretion and Elimination of Cardiac    Natriuretic Peptides During Acute Volume Overload. Hypertension    2000; 36: 355-359.-   6. Richards A M, Nicholls M G, Troughton R W, Lainchbury J G,    Elliott J, Frampton C, Espiner E A, Crozier I G, Yandle T G,    Turner J. Antecedent hypertension and heart failure after myocardial    infarction. J. Am. Coll. Cardiology. 2002 39: 1182-1188.-   7. Troughton R W, Prior D L, Pereira J J, Martin M, Fogarty A,    Morehead A, Yandle T G, Richards A M, Starling R C, Young J B,    Thomas J D, Klein A L. Plasma B-type natriuretic peptide levels in    systolic heart failure: importance of left ventricular diastolic    function and right ventricular systolic function. J Am Coll Cardiol.    2004 43:416-422.-   8. Troughton R W, Frampton C M, Yandle T G, Espiner E A, Nicholls M    G, Richards A M. Treatment of heart failure guided by plasma    amino-terminal brain natriuretic peptide (N-BNP) concentrations.    Lancet 2000 355: 1126.1130.-   9. Multiple Sequence Alignment with the Clustal series of programs    Nucleic Acids Res (2003) 31 (13): 3497-500.-   10. Bowie, J. U et al., (1990). Decipeing the message in Protein    Sequences: Tolerance to Amino Acid Substitutions. Science 247,    1306-1310.-   11. Harbour and Lane 1998. Antibodies: A Laboratory Manual, Cold    Spring Harbour Press New York.²⁷-   12. Kohler and Milstein 1975. continuous Cultures of Fused Cells    Secreting Antibody of Predefined Specficity. Nature, 256, 495-497.-   13. Verhoeyen M. C Milstein, and G Winter Reshaping human    antibodies: grafting an antilysozyme activity. Science 1988 Mar. 25;    239(4847):1534-6.-   14. Jones, P. T., Dear, P. H., Foote, J., Neuberger, M. S. and    Winter, G. “Replacing the complementarity-determining regions in a    human antibody with those from a mouse.” Nature (1986) 321: 522-525.-   15. Riechmann L, Clark M, Waldmann H, Winter G. Reshaping human    antibodies for therapy. Nature. 1988 Mar. 24; 332(6162):323-7.-   16. Hoogenboom H R, Winter G (1992) Human antibodies from synthetic    repertoires of germline VH gene segments rearranged in vitro. J Mol    Biol. 1992 Sep. 20; 227 (2):381-8.-   17. Michael Neuberger (1996) Generating high-avidity human Mabs in    mice Nature Biotechnology 14, 826-   18. Tristan J. Vaughan, Jane K. Osbourn & Philip R. Tempest (1998)    Human antibodies by design. Nature Biotechnology 16, 535-539-   19. Milstein and Cuello (1983) The co-expression of two    immunoglobulin heavy-chain/light-chain pairs, where the two heavy    chains have different specificities, Nature, 305:537-539.-   20. Suresh, M. R., Cuello, A. C. and Milstein, C. (1986) Bi-specific    monoclonal antibodies from hybrid hybridomas. Methods in Enzymology,    121: 210-228.-   21. Brennan et al., “Preparation of bispecific antibodies by    chemical recombination of monoclonal immunoglobulin G1 fragments”    Science 229:81-83 (1985).-   22. Hunt P J, Richards A M, Nicholls M G, Yandle T G, Doughty R N,    Espiner E A. Immunoreactive amino terminal pro brain natriuretic    peptide (NT-proBNP): a new marker of cardiac impairment. Clin.    Endocrinol. 1997 47:287-296.-   23. The Immunoassay Handbook. 3^(rd) edition, ed. David Wild.    Elsevier Ltd, 2005.-   24. Solber H. Approved recommendation (1987) on the theory of    reference values. Part 5. Statistical treatment of collected    reference values. Determination of reference limits. Journal of    clinical Chemistry and Cilinical Biochemistry 1987 25:645-656.-   25. Braud V M, Allan D S, O'Callaghan C A, Soderstrom K, D'Andrea A,    Ogg G S, Lazetic S, Young N T, Bell J I, Phillips J H, Lanier L L,    McMichael A J. HLA-E binds to natural killer cell receptors    CD94/NKG2A, B and C. Nature 1998 391:795-799.-   26. Universal definition of myocardial infarction. Consensus    statement from the Joint ESC/ACCF/AHA/WHF Taskforce for the    redefinition of myocardial infarction. Circulation 2007    116:2634-2653.-   27. National Academy of Clinical Biochemistry and IFCC Committee for    standardisation of markers of cardiac damage laboratory medicine    practice guidelines: analytical issues for biochemical markers of    acute coronary syndromes. Circulation 2007 115:e352-e355.-   28. Kunkel, Thomas A. Rapid and efficient site-specific mutagenesis    without phenotypic selection. Proc. Natl. Acad. Sci. USA Vol. 82,    pp. 488-492, January 1985.-   29. Techniques in Protein Modification By Roger L. Lundblad Edition:    2 Published by CRC Press, 1995 288 pages.-   30. Atherton et al. (1989) Solid Phase Synthesis: a practical    approach, IRL press.-   31. Skyler J S. Non-insulin-dependent diabetes mellitus: a clinical    strategy. Diabetes Care. 1984 May-June; 7 Suppl 1L118-29.

The present invention is not limited by the aforementioned particularpreferred embodiments. It will occur to those ordinarily skilled in theart that various modifications may be made to the disclosed preferredembodiments with-out diverting from the concept of the invention. Allsuch modifications are intended to be within the scope of the presentinvention.

All patents, publications, scientific articles, web sites, and otherdocuments and materials referenced or mentioned herein are indicative ofthe levels of skill of those skilled in the art to which the inventionpertains, and each such referenced document and material is herebyincorporated by reference to the same extent as if it had beenincorporated by reference in its entirety individually or set forthherein in its entirety. Applicants reserve the right to physicallyincorporate into this specification any and all materials andinformation from any such patents, publications, scientific articles,web sites, electronically available information, and other referencedmaterials or documents.

The written description portion of this patent includes all claims.Furthermore, all claims, including all original claims as well as allclaims from any and all priority documents, are hereby incorporated byreference in their entirety into the written description portion of thespecification, and Applicants reserve the right to physicallyincorporate into the written description or any other portion of theapplication, any and all such claims. Thus, for example, under nocircumstances may the patent be interpreted as allegedly not providing awritten description for a claim on the assertion that the precisewording of the claim is not set forth in haec verba in writtendescription portion of the patent.

All of the features disclosed in this specification may be combined inany combination. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Thus,from the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for the purposeof illustration, various modifications may be made without deviatingfrom the spirit and scope of the invention. Other aspects, advantages,and modifications are within the scope of the following claims and thepresent invention is not limited except as by the appended claims.

The specific methods and compositions described herein arerepresentative of preferred embodiments and are exemplary and notintended as limitations on the scope of the invention. Other objects,aspects, and embodiments will occur to those skilled in the art uponconsideration of this specification, and are encompassed within thespirit of the invention as defined by the scope of the claims. It willbe readily apparent to one skilled in the art that varying substitutionsand modifications may be made to the invention disclosed herein withoutdeparting from the scope and spirit of the invention. The inventionillustratively described herein suitably may be practiced in the absenceof any element or elements, or limitation or limitations, which is notspecifically disclosed herein as essential. Thus, for example, in eachinstance herein, in embodiments or examples of the present invention,the terms “comprising”, “including”, “containing”, etc. are to be readexpansively and without limitation. The methods and processesillustratively described herein suitably may be practiced in differingorders of steps, and that they are not necessarily restricted to theorders of steps indicated herein or in the claims.

The terms and expressions that have been employed are used as terms ofdescription and not of limitation, and there is no intent in the use ofsuch terms and expressions to exclude any equivalent of the featuresshown and described or portions thereof, but it is recognized thatvarious modifications are possible within the scope of the invention asclaimed. Thus, it will be understood that although the present inventionhas been specifically disclosed by various embodiments and/or preferredembodiments and optional features, any and all modifications andvariations of the concepts herein disclosed that may be resorted to bythose skilled in the art are considered to be within the scope of thisinvention as defined by the appended claims.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

It is also to be understood that as used herein and in the appendedclaims, the singular forms “a,” “an,” and “the” include plural referenceunless the context clearly dictates otherwise, the term “X and/or Y”means “X” or “Y” or both “X” and “Y”, and the letter “s” following anoun designates both the plural and singular forms of that noun. Inaddition, where features or aspects of the invention are described interms of Markush groups, it is intended, and those skilled in the artwill recognize, that the invention embraces and is also therebydescribed in terms of any individual member and any subgroup of membersof the Markush group, and applicants reserve the right to revise theapplication or claims to refer specifically to any individual member orany subgroup of members of the Markush group.

Other embodiments are within the following claims. The patent may not beinterpreted to be limited to the specific examples or embodiments ormethods specifically and/or expressly disclosed herein. Under nocircumstances may the patent be interpreted to be limited by anystatement made by any Examiner or any other official or employee of aPatent Office unless such statement is specifically and withoutqualification or reservation expressly adopted in a responsive writingby Applicants.

The invention claimed is:
 1. A method for diagnosing or monitoring diabetes in a subject, the method comprising: (a) measuring the level of an insulin signal peptide (INS-SP) fragment defined by INS-SP (1-9) SEQ ID NO:16 in a biological sample from the subject; and (b) comparing the level of INS-SP fragment with the INS-SP fragment level from a control, wherein a measured level of INS-SP fragment lower than the control level is indicative of diabetes.
 2. A method for diagnosing or monitoring an acute cardiac disorder (ACD) in a subject, the method comprising: (a) measuring the level of on insulin signal peptide (INS-SP) fragment defined by INS-SP (1-9) SEQ ID NO:16 in a biological sample from the subject; and (b) comparing the level of said INS-SP fragment with the INS-SP fragment level from a control, wherein a measured level of INS-SP fragment higher than the control level is indicative of ACD.
 3. A method of claim 2, wherein said method is used to monitor a response to treatment of an acute cardiac disorder (ACD) in a subject, wherein a change in the measured level of INS-SP fragment from the control level is indicative of a response to the treatment.
 4. A method of claim 2, the method comprising measuring the level of INS-SP fragment in a biological sample from the subject within the first six hours of onset of, or clinical presentation with, acute cardiac disorder (ACD).
 5. A method according to claim 2, wherein the level of INS-SP fragment is measured within the first hour of onset of ACD, or clinical presentation with, acute cardiac disorder (ACD).
 6. A method according to claim 2, wherein a level of INS-SP fragment in the sample in the range 40 to 250 pmol/L, is indicative of ACD.
 7. A method according to claim 2, wherein a level of INS-SP fragment in the sample which is 5 to 15 times higher than the control level is indicative of ACD.
 8. A method according to claim 2, wherein the acute cardiac disorder is an acute myocardial infarction (AMI) with ST-elevation on presenting ECG, unstable angina, an acute non ST-elevated myocardial infarction; cardiac ischemia, acute cardiac injury, acute cardiac damage resulting from acute drug toxicity, an acute cardiomyopathy, or a cardiac transplant rejection episode.
 9. A method according to claim 2, wherein the biological sample is a blood, plasma, serum, saliva, interstitial fluid, urine or heart tissue sample.
 10. A method according to claim 2, wherein the measuring step comprises (a) binding INS-SP fragment with a binding agent; and (b) measuring the level of bound INS-SP fragment.
 11. A method according to claim 2, wherein the binding agent is a binding agent that specifically binds INS-SP (1-9) (SEQ ID NO:16).
 12. A method according to claim 2, wherein the level of INS-SP fragment is measured using an assay selected from mass spectroscopy (including SELDI, ESI, MALDI or FTIC), RIA, ELISA, fluoroimmunoassay, immunofluorometric assay, and immunoradiometric assay.
 13. A method according to claim 2, which further comprises measuring the level of one or more non-INS-SP fragment markers of said ACD, and comparing the levels against marker levels from a control wherein a deviation in the measured level from the control level, together with a measured level of INS-SP fragment which is higher than the control level of INS-SP fragment, is predictive or diagnostic of the ACD, or can be used to monitor said ACD.
 14. A method as claimed in claim 13 wherein the non-INS-SP fragment markers are selected from the group consisting of troponin T, troponin I, creatine kinase-MB, myoglobin, ANP, ANP-SP, BNP, NT-BNP, BNP-SP, LDH, aspartate aminotransferase, H-FABP, endothelin, adrenomedullin, renin, ischemia modified albumin and angiotensin II.
 15. A method according to claim 1, which further comprises measuring the level of one or more non-INS-SP fragment markers of diabetes and comparing the levels against marker levels from a control wherein a deviation in the measured level from the control level of non-INS-SP fragment marker, together with a measured level of INS-SP fragment which is lower than the control level of INS-SP fragment, is predictive or diagnostic of diabetes or can be used to monitor diabetes.
 16. A method as claimed in claim 15 wherein the non-INS-SP fragment markers are selected from the group consisting of glucose, insulin, lactate, trigyclerides and fatty acids or markers therefor.
 17. The method of claim 2 wherein measuring the level of the INS-SP fragment is performed using an antibody or antigen-binding fragment of the antibody, which antibody or antigen-binding fragment selectively binds INS-SP (1-9) SEQ ID NO:16.
 18. A method according to claim 1, wherein said method is used to monitor a response to treatment of diabetes in a subject, wherein a change in the measured level of INS-SP fragment from the control level is indicative of a response to the treatment.
 19. A method according to claim 1, wherein the biological sample is a blood, plasma, serum, saliva, interstitial fluid, or urine.
 20. A method according to claim 1, wherein the measuring step comprises (a) binding INS-SP fragment with a binding agent; and (b) measuring the level of bound INS-SP fragment.
 21. A method according to claim 20, wherein the binding agent is a binding agent that specifically binds INS-SP (1-9) (SEQ ID NO:16).
 22. A method according to claim 1, wherein the level of INS-SP fragment is measured using an assay selected from mass spectroscopy (including SELDI, ESI, MALDI or FTIC), RIA, ELISA, fluoroimmunoassay, immunofluorometric assay, and immunoradiometric assay.
 23. The method of claim 1, wherein measuring the level of the INS-SP fragment is performed using an antibody or antigen-binding fragment of the antibody, which antibody or antigen-binding fragment selectively binds INS-SP (1-9) SEQ ID NO:16. 